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Adult Stem Cell-Based Gene Therapy For Alpha 1-Antitrypsin Deficiency

Permanent Link: http://ufdc.ufl.edu/UFE0024912/00001

Material Information

Title: Adult Stem Cell-Based Gene Therapy For Alpha 1-Antitrypsin Deficiency
Physical Description: 1 online resource (130 p.)
Language: english
Creator: Li, Hong
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: 1, adeno, adipose, adult, alpha, antitrypsin, associated, bone, cell, deficiency, derived, hepatic, lentiviral, marrow, mesenchymal, oval, stem, tissue, vector, virus
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Adult Stem Cell-Based Gene Therapy For Alpha 1-Antitrypsin Deficiency 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 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.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Hong Li.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Song, Sihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024912:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024912/00001

Material Information

Title: Adult Stem Cell-Based Gene Therapy For Alpha 1-Antitrypsin Deficiency
Physical Description: 1 online resource (130 p.)
Language: english
Creator: Li, Hong
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: 1, adeno, adipose, adult, alpha, antitrypsin, associated, bone, cell, deficiency, derived, hepatic, lentiviral, marrow, mesenchymal, oval, stem, tissue, vector, virus
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Adult Stem Cell-Based Gene Therapy For Alpha 1-Antitrypsin Deficiency 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 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.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Hong Li.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Song, Sihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024912:00001


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1 A D ULT 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

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2 2009 Hong Li

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3 To my parents, Guoji Li and Baoz hu Zheng, and my sister, Xia Li

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4 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 problem s and guided me development as a scie ntist. 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 val uable 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 h ow 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 a ssistance with immunohistochemistry assays. Also many thanks go out to all the members of Dr.Songs lab, in particular, Dr.Christian Grimstein and Matthias Fueth, who s upported me in all my endeavors Last, but not the least, I would like to thank my pare nts, 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. S he 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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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF FIGURES .............................................................................................................................. 8 ABSTRACT ........................................................................................................................................ 10 CHA PTER 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 2 MATERIALS AND METHOD S ............................................................................................... 48

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6 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 Transducti on ................................................................................................................... 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 Osteogen e sis ......................................................................................................................... 52 Liver Directed Transplantation of Adult Stem Cell s ................................................................ 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 LIV ER 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 4 EX VIVO TRANSDUCTION AND TRA NSPLANTATION 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 Mar row Cells ................................... 73 Bone Marrow Cell Transplantation Resulted in Sustained Levels of hAAT in Recipient Circulation ....................................................................................................... 74 Discussion .................................................................................................................................... 75 5 ADIPOSE TISSUE DERIVED MESENCHYMAL STEM CELL BASED LIVER GENE DELIVERY ..................................................................................................................... 86 Summary ...................................................................................................................................... 86 Introduction ................................................................................................................................. 87 Experimental Design ................................................................................................................... 89

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7 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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8 LIST OF FIGURES Figure page 2 1 rAAV vectors construct ........................................................................................................ 57 3 1 Experimental outline of oval ce ll study.. .............................................................................. 64 3 2 Flow cytometric quantification of green fluorescent oval cells after transduction of rAAV-CB GFP vectors. ......................................................................................................... 65 3 3 Const ruct of Lenti -CB -hAAT vectors. ................................................................................ 66 3 4 Ex vivo transduction of oval cells.. ........................................................................................ 67 3 5 Detection of expression of hAAT in re cipient liver after transplantation of ex vivo transduced oval cells by immunostaining. ............................................................................ 68 3 6 hAAT expressed from engrafted oval cells.. ........................................................................ 69 4 1 Experimental Outline of BM cell study .. .............................................................................. 78 4 2 Ex vivo transduction of BM cells. ......................................................................................... 79 4 3 Detection of expression of human alpha1 antitrypsin (hAAT) in recipient liver after transplantation of viral vector infected BM cells by immunostaining.. .............................. 80 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). ........................................................................................... 81 4 5 Detection of donor cells in recipient liver after BM cell tr ansplantation by fluorescence in situ hybridizations (FISH) for Y -chromosome. ......................................... 82 4 6 Detection of coexpression of human alpha 1 antitypsin (hAAT) and mouse albumin by immunostaining.. ............................................................................................................... 83 4 7 Multi -ogran homing of transplanted BM cells.. ................................................................... 84 4 8 Detection of expression of human alpha1 antitrypsin (hAAT) in the recipient serum.. ... 85 5 1 Experimental outline of AT -MSC study .............................................................................. 96 5 2 Characterization of AT -MSCs. .............................................................................................. 97 5 3 In vivo muscle or liver transduction by ssAAV and dsAAV vectors.. ............................... 98 5 4 Ex vivo AT MSCs transduction efficiency of rAAV vectors.. ............................................ 99

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9 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.. ........... 100 5 6 Detection of donor cells in recipient liver after AT -MSCs transplantation by fluorescence in situ hybridizations for Y -chromosme. ...................................................... 101 5 7 Detection of coexpression of human alpha 1 antitypsin (hAAT) and mouse albumin by immunostaining.. ............................................................................................................. 102 5 8 Multi -ogran homing of transplanted AT -MSCs.. ............................................................... 103 5 9 Detectio n of expression of human alpha 1 antitrypsin (hAAT) in the serum.. ................ 104

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10 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 h uman 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 t h ree 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. Usi ng oval cells, we confirmed the feasibility of r ecombinant adeno associated virus (r AAV ) vector mediated ex vivo transduction and transplantation. Considering isolation of oval cell for autologous transplantation is not clinically applicable, we ha ve test the use of BM cells. We showed that both lentiviral vector and rAAV vectors can transduce BM cells. Transplantation of

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11 transduced BM cells sho wed that BM cells transdifferentiated into hepatocytes and mediated transgene (hAAT) expression in the live r. Impo rtantly, 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 e fficiently 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 hAA T 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 the rapeutic levels of transgene expression, and gene correction in AT -MSCs for AAT deficiency associated liver disease

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12 CHAPTER 1 LITERATURE REVIEW Introduction Alpha 1 antitrypsin (AAT) deficiency is a n autosomal recessive condition caused mostly by a sin gle gene mutation in the AAT coding sequence. This mutation results in abnormal aggregation of AAT protein in hepatocytes and consequent absence of AAT pro tein in the systemic circulation which leads to hepatocyte dysfunction or lung destruction. Adult st em 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 cell s 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 po tential of a therapy for AAT deficiency by using adult stem cells, this chapter review s 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 suppo rt for the adult stem cell -based gene therapy. Alpha 1 Antitrypsin D eficiency AAT deficiency is a genetic disorder resulting from mutation s of AAT gene. The mutation results in a red uction of serum levels of alpha 1 antitrypsin and consequently an increase d 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 w ith severe AAT deficiency, but fewer than 10 percent have been diagnosed .3

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13 AAT B iology AAT, a serine protease inhibitor, is mainly synt he sized by hepatocytes and secreted into the circulatory system with a serum concentration ranging from 20 to 53 M It is the second abundant pl a sma protein next to albumin and has a serum half -life of 4 to 5 days.2 The AAT molecule is a 52kD glycoprotein with 394residue and three asparagines -linked carbohydrate side chains.46 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 com ponent is elastin against the excessive proteolytic damage of neutrophil elastase (NE) by combining the AAT inhibitory site (Met358Ser359) with the active proteolytic site (catalytic triad Ser173His41-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, design ated 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 vari ant); deficient variants, with

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14 reduced but detectable AAT serum concentration and associated wit h lung and/or liver disease, (i.e Z and S variants ); dysfunctional variants associated with altered function, (i.e. AAT Pittsburgh, a thrombin inhibitor inst ead of a neutrophil elastase inhibitor ); and null variants, with no serum AAT and an increased risk of lung disease.11, 12 AAT D eficiency P athogenesis AAT deficiency was discovered in 1963 by C. -B. Laurell and S.Eriksson in Malm Sweden13 when they described that three of five individuals lacking an alpha 1 -globulin band in electrophoretic analysis of serum had significant pulmonary dise ase. It was known that about 90% of the alpha 1 globulin band was a single protein capable of i nhibiting the proteolytic action of trypsin; hence, the term alpha 1 antitrypsin deficiency was used to d efine 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 tryp sin, 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 1 antitrypsin 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 ar e the two most common mutated allele s The Z allele contains a G to -A mutation within exon V which results in a change from a negatively charged glutama te (GAG) at position 342 to a positively charged lysine (AAG) within the protein. This amino acid substitution causes abnormal folding and the accu mulation of AAT within the endoplasmic reticulum. The Z mutation accounts for >95% of all AAT deficiency alleles.14 I ndivid uals 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

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15 variant is a n 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 Individual s with two S alleles have a 40% decrease in plasma AAT concentrations .13 Consequently, AAT plasma concentration s in both ZZ homozygotes and SZ heterozygotes are insufficient to ensure the lifetime protection of tissue s from the proteolytic damage of NE and cause cli nical deficiency of AAT. Compound heterozyg otes for Z allele and S allele are also at risk if the AAT serum levels fall below the protective threshold of 11 M .16, 17 AAT deficiency is more prevalent in Caucasians, a l though 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 prote ase within the lung, which explains why AAT def iciency 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 w hom symptom will present by age 35 to 50 years .21, 22 In contrast, n on -AAT deficient individuals typically do nt 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 individual s 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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16 15years.23 Oxidants, particularly oxidants in cigarette smoke can easily oxidize the Met358, the active in hibitory 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 T he 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 i s likely due to the polymerization of AAT protein, a result of a change in t he three -dimensional structure secondary to the Glu342 .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 differe nt from that of AAT deficiency related lung disease. Only approximately 10 15% of AAT deficient individual s with two homozygous Z alleles w ill develop the liver disease23, while lung disease will occur in any AAT deficient individual with a serum AAT level of < 11 M The liver disease presents in i nf ancy 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 indiv idual.25 T here is no available therapy for AAT d eficiency -related liver disease o th er than liver transplantation. Nevertheless, liver transplantation has a number of important drawbacks, including shortage of donors, lifelong immunosuppressive therapy and a significant mo rtality rate. Current Therapy for AAT D eficiency The prevention of lung disease in AAT deficiency is a relatively straightforwar d concept, since the serum levels at 11M or 800g/ml and ab ove is a clear indicator of having restored anti -neutrophil elastase defense.23 Protein replacement therapy is the only FDA approved treatment for individuals wit h emphysema due to AAT deficiency. The regimen of once -weekly

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17 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 M 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 donors plasma from which AAT protein is made. Fortunately, there has been no reported cases of blood born disease s (HIV or hepatitis) linked to receiving this therapy. Most common reported si de 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 pro duction 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 so ught to address the shortage of AAT protein. A yeast derived recombinant AAT (rA AT) and a transgenic sheep/goat derived AAT product have been generated and tested in clinical trial s for safety and efficacy.27 31 Unl ike human AAT protein, these rAAT products derived from yeast and transgenic animal s have different glycosylation. For example, yeast -derived rAAT lacks carbohydrate side chains 29, 32 and thus has a high renal clea rance and a much shorter plasma half -life than natural human AAT protein, which make s 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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18 route of administration also reduce the dose of AAT protein in replacement therapy. The protective threshold level in the lung is 1.2M, 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 twothirds in the amount.33 Unfortunately, th is 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 p rotein replacement strategies. Besides avoiding t he 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 rang e of fluctuations in AAT levels that is seen in protein replacement therapy. Most important ly, gene therapy can provide a potential treatment for AAT deficiency associated liver disease by implementing the strategy of delivering shRNA to correct or block t he m utant AAT gene in hepatocytes. Although the costliness of gene therapy often comes into question, protein replacement therapy is also very expe nsive. The mean annual cost of protein therapy was from $ 30,000 to $40,000, bas ed 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 system s (e.g. cationic liposome, naked DNA injection, and gene particle bombardment delivery ), and the viral delivery system s (e.g. adenoviral retroviral and Adenoassociated vir al 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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19 biologically active AAT into serum.23 A phase I clinical trial evolving the intramuscular injection of recombinant adeno associated virus serotype 2 alpha 1antitrypsin (rAAV2 -AAT) vector in 12 AAT deficiency adults has been done. Four dose cohorts ranging from 2.11012 ve ctor genome (VG) to 6.91013 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 6.91013 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 therapeut ic 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 effi ciency advantage of hundred-fold over rAAV2 in mouse muscle.37 Recombinant AAV V ector AAV G enome Adenoassociated virus (AAV) is a non pathogenic DNA parvovirus with a linear single stranded 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 repeat s (ITR s ) that flan k 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 site specific, 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

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20 integration locus on human chromosome 19 to regulate viral transcripti on 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 i nto 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 alterna tive 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 m olar ratio of 1:1:1 0.46 The ITR is the onl y 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 T shaped hairpin structure and the other 20 bases, call ed 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, tra fficking to the nuclear, uncoating, and conversion of single -stranded (ss) AAV genome to double stranded (ds) molecule. AAV2 gains entry into target cell by binding to primary attachment receptor heparin su lphate proteoglycans (HSPG).51 F ibroblast growth factor receptor 1 (FGFR1)52V5 integrin heterodimers ,53 and hepatocyte gr owth factor receptor (HGFR)54 serve as co receptors to facilitate the internalization. AAV2 internalizes rapidly by c lathrin -mediated endocytosis from clathrincoated pits (half -time <10 min).55 For successful infection, AAV particle need to escape from endosome. Acidic pH in the endosome is required to induce the conformational changes of the key capsi d subunits necessary f or priming the virus for endosom al release.46, 56 The phospholipase A2 (PLA2) domain located at the N -terminus of VP1 is exposured during endosomal process. PLA2 domain is conserved in parvovir uses and has been shown to play an important role during AAV trafficking, possibly helping AAV escape the late

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21 endosome.46 Point mutation in PLA2 domain results in delay onset and low level of transgene expression.57 T he ubiquitin-proteasome pathway impairs AAV intracellular trafficking.58 Recent research demonstrated that mutat ing the surface exposed tyrosine residues on AAV2 capsid wo uld help viral vector circumvent the ubiquitination step and thus avoid proteasome -mediated degradation which resulted in increased transduction efficiency in both in vitro and in vivo experiments.59 Release of the virus into the cytosol occurs within 30min postinfection.55 Following endosomal escape, AAV accumulates around the nucleus by 2 h following internalization and slowly penetrates through the nuclear pore complex (NPC) into the nucleus.55 In the absence of adenovirus, AAV particles persist perinuclearly for 16 24 h. Few, if any, intact AAVcapsids were found in the nucleus. In the presence of adenovirus, cytoplasmic AAV quickly translocate into nucleus as intact particle as early as 40 min after coinfection.60 Whether AAV uncoating take places before or after nuclear entry is not clear. After uncoating, ssAAV genome was release. The ss -genome has one of two fates: either it convert s to the stable bio logically active ds genome by anneal ing to another complementar y ss -genome or synthesizing second strand utilizing the cellular factors, or it is targeted by cellular protein, e.g. FKBP52 binds to the AAV ITR and inhibit the second -strand synthesis, for degradation.6163 Resear ch also has shown the rapid uncoating of vector genome is the key to efficient liver transduction with AAV8 pseudotype vector, AAV2 -based vector ge n ome packaged inside AAV8 capsid since the ss genome are more likely to process along the annealing pathway than along the pathway that leads to degradation.64 The efficiency of AAV transduction depend on the efficiency at each step of AAV infection, among which nuclear slow translocation, slow uncoating and limited secondstrand synthes is are the rate -limiting step. This explains why AAV vector demonstrates slow rise

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22 in gene expression, e.g. AAV2 transgene expression is characterized by a lag phase of up to 6 weeks.64 AAV S erotype AAV serology is defined as the inability of an antibody that is reactive to the viral capsid proteins of one serotype in neutralizing those of another serotype.65 Therefore, a new serotype can only be named when newly isolated virus has been tested for neutralization against all ex isting and characterized serotypes. If there is no serological difference from any existing serotype, newly isolated virus will be named as the variant of the corresponding serotype.65 To date, total 12 AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or from human/nonhuman primate tissues6672 since AAV serotype 2 the first AAV serotype, was discovered as a contaminant in an adenovirus type 12 stock in the late 1960s .67 AAV serotypes display distinct cell and tissue affinities attributed to the physical compositions of their capsid protein coa t. For example, AAV2 transduces a wide range of tissu e with moderate efficiency including liver, muscle, lung and central nervous system tissue. AAV serotypes 1, 5, 8, and 9 are known to perform high level transductions of skeletal muscle, retina, liver, and heart respectively. AAV serotype s 6 and 7 are als o prone for skeletal muscle transduction. AAV9 exhibits a similar profile to AAV8 with capability of transducing liver, heart, skeletal muscle and pancreatic acinar cells. The interaction of the AAV capsid with cell surface receptors initiate the first ste p of AAV infection, cell surface binding. The cell surface receptors have been identified for some of the AAV serotypes. Unlike AAV2, AAV4 and AAV5, which display different tropism with respect to AAV2, use O and N linked sialic acids, respectivel y, for c ell surface binding.73 T he platelet -derived growth factor receptor also facilitates AAV5 entry as a co receptor .74 In addition, a 37 kDa/67kDa laminin rece ptor has been identi fied as a receptor for

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23 AAV serotype 2, 3, 8, and 9.75 Cell surface receptors utilized for cell binding by other AAV serotypes remain to be determined. Broad tissue tropisms of different AAV serotypes make AAV vector as a promising gene delivery tool, but this might also lead to nonspecific targeting on cell where it could be deleterious, or simply dilute the vector dose in tissue where it is not useful, especially systemic delivery of AAV vectors. Modifying virus cap sid protein, e.g. transcapsidation, adsorption modification, mosaic capsid, and chimeric capsid is one of approaches to develop naturally occurring AAV serotypes into clinical applicable vectors,65 since virus capsid is responsible for binding to c ell surface receptors. Transcapsidation is packaging the genome containing ITRs from one serotype into the capsid of another serotype. For instance, AAV2 ITR has been cross packaged into AAV1 capsid and tested in preclinical trial for muscle directed gene therap y for alpha 1 antitrypsin deficiency37 because rAAV1 vectors hav e shown hundred-fold more potency for murine muscle tran sduction than rAAV2 vectors .76 A drawback of this strategy is that AAV2 ITR-containing genome can not be packaged into AAV5 capsid due to only 60% similarity between those two serotypes of Rep and ITR genes.77, 78 AAV5 Rep protein recognize and cleavage distinct TRS sequence which is absent from the ITRs of other AAV serotypes.77 There are still some cell types that are non -permissive to any of these AAV serotypes, and thu s transcapsidation wont improve the transduction efficiency. Adsorption of receptor ligands to 2 antibody,79 that reacts with the viral capsid as well as a cellular receptor and form a conjugate ideally able to retarget virus to a refra ctory cell type such as megakaryocyte cells. A mosaic capsid AAV is a virion composed of a mixture of virus capsid protein from different serotypes.65 For instance, a mosaic virus was generated by using a mixture of AAV helper plasmids encoding both AAV1 and AAV2 capsid in

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24 the transfection process. The resulting mosaic virus possesses the advantage of each parental serotype so that it can be purified by heparin column and has similar transduction efficiency to those of AAV1 in muscle or AAV2 in liver ; that is, it combines the best transduction characteristics of both parental AAV serotypes.80 Chimeric capsid AAV can be defined as an insertion of a foreign protein or peptide sequence into the open reading frame of the capsid gene as a ligand to a receptor expressed on the target cell type. VP2 N terminus is by far the best terminal position for epitope insertion without interrupting the efficiency of viral infection.65 AAV2 heparin binding domain (HBD) consists of a total of four arginine (R) residues and one lysine residue with R585 and R587 representing the most crucial component.81, 82 Insertion of 7 amino acid peptide into this region have shown to be able to ablate the HSPG binding ability and allow retargeting of the virus to bind to the receptor of interest.83 The search for a position that can tolerate peptide insertion and yet be able to retain most of the biology activity of AAV was difficult and tedious. DNA family shuf fling has also been introduced into the realm of AAV vector evolution. The basic concept of this technology is the in vitro recombination of related parental gene with >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 th is 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 -C omplementary 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 ex pression 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

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25 conversion of ssAAV genome to dsAAV genome either through host -cell DN A 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 TR S 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 self complementary nature with a covalently closed ITR at one end and two open-end ed 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, He la, and COS 7 cells show dsAAV2 CMV GFP vector yielded 10 20% green fluorescent cells one day after infectio n 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 a t 5105 MOI, but less than 5 % hepatocytes were permissive to conventional ssAAV2 vector for longterm 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 b e 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 drugresistant phenotype.88 Whats mor e, 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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26 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 AAV 2 infects a human cell alone, its gene expression is autorepressed and establishes laten cy 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 organiz ation.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 separ ated 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 A AV 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 AA VS1 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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27 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 extrachr omosomal, 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 (<0.5%). The integrations are associated with chromosomal deletions of up to 2kb at integration sites and occur preferentially in actively transcribed genes (e.g., 72% into genes, 28% in intergenic region) in mice.98, 99 Non -homologous endjoining (NHEJ) has been proposed to be the possible machinery that rAAV vectors use to integrate at chromosome double -strand breaks (DSBs).99 One potential problems of integration is inactivation of a chromosomal gene, but in m ost cases an intact copy would still function on the other autosomal chromosome. Activating an oncogene is the major concern of host genome insertion. That rAAV vectors prefer actively transcribed as opposed to silent gene does reduce this risk, as the gen es will already be expressed. However, insertional mutagenesis by rAAV vector was observed recently in the development of hepatocellul ar carcinoma in mice.100 This finding raises s afety concerns on the clinical appl ication of AAV vectors. To address this problem, site -specific integration rAAV vector is under investigation since wild type AAV does integrate into human chromosome 19 which has been shown to be safe integration site.46 Zhang et al (2007) designed a bipartite rAAV vector composed of a vector enc oding AAV rep gene driven by the simian virus 40 early promoter rather than the P5 promoter, and a second vector containing P5 IEE plus the GFP reporter gene.101 Co infecting Hela cells with these two vectors results in >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, S m i th et al (2008) has

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28 shown robust and persis tent 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 electr oporation or Lipofectamine 2000. 4.16% of hESC clones achieved AAVS1-targeted integration. The AAVS1 targeted hESCs retained their phenotypes and differentiated into all three primary germ layers. EGFP expression from AAVS1 -targeted clones showed sig nificantly 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 cel l 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 V ector and Its Application for Stem C ell Transduction The first infectious clone of AA V2 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, Parkinsons disease, muscul ar 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 AAV2based 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 fal l short of requirement for adequate and organ -

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29 specific transgene expression. As a result, ongoing research efforts are focused on developing new r AAV vector by modifying bot h AAV genome and capsid protein as described above. Another direction of future the rapy is the combination of rAAV -mediated gene delivery with stem cell therapy. The capability of self renewal and differentiation of stem cells make s them to be the promising target of virus vector for long term gene correction. Although AAV vectors can tr ansduce a broad range of tissues and cel ls, 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 r AAV2 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+CD38cell population.108 In addition, CD34+CD38and CD34+G0cells, 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 hum an 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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30 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 reache d 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 i nduce 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 appr oaches 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 V ector Lentivirus is a member of retrovirus, a RNA virus family, of which particle consists of 2 identical single -stra nded RNA molecules .113 A unifying feature of these viruses is that replication involve s the process of conversion of the viral RNA genome into double -stranded DNA, hence the designat ion retro (backward) virus .114 Lentivirus is unique among retrovirus

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31 because of its ability to infect nondividing and ter minally differentiated cells such as HSCs and neurons, something that other retroviruses cannot do .115 The ab ility 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 immunodefi ciency virus (HIV 1and2)116118, simian i mmunodeficiency virus (SIV)119, feline immunodeficiency virus (FIV)120 122, equine infectious anemia vi rus (EIAV)123, 124, caprine arthriti s encephalitis virus (CAEV)125, and bovine immu nodeficiency virus (BIV)126, 127. Among those, the HIV 1 based lentiviral vectors are prototypical and predominan tly 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 (C A), 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 p 6. 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, wh ich 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 tw o additional regulatory genes, tat and rev and four accessory genes, vif vpu vpr and nef all of which are involve d 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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32 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 cotranfecting 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 HI V 1 infection129, thalassaemia130, sickle cell disease131, Parkinsons 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-ba se antisense gene against the HIV envelope. VRX496 directly interferes with wildtype HIV (wt HIV) expression via anti -env antisense expression in vector transduced CD4+ T cells that become infected with wtHIV 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 stabl e, 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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33 Adult Stem C ells 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 tissue s where they are found. Regenerative medicine devotes to rebuilding damaged organs from stem cells includ ing cloned cells, embryonic or fetal stem cells, or adult stem cells Of all the different stem cell types, o nly adult stem cells might provide more medical solutions because of avoiding the ethical and legal problems of cloni ng 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 Cell s Hepatic o val cell s are bipotential progenitor s that can differentiate into two types of epithelial cells within the liver, hepatocyte s and bile ductular cell s, 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 I solation of Oval C ell Oval cells were first described by K inosita 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 hepa tocytes), and large nucleus to cytoplasm ratio.140 Oval cells didnt invoke attention until Thorgeirsson et al. show ed that oval cell s can differentiate into hepatocytes in the

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34 1980s.141, 142 Under normal condition s oval cells a re quiescent and reside in the C anals of Hering (also called cholangioles, terminal ducts, or ductules).143, 144 When sever e liver damage occur s and the proliferation of hepatocytes is blocked by exposure to hepatotoxins or carcinogens, oval cells are acti vated, 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 model s 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 compartmen t doesnt response to this two-step induction protocol used in the rat model. Instead, mouse was placed on a diet containing the chemical 3, 5 diethoxycarbonyl 1, 4 -dihydrpcollodine (DDC) at 0.1% concentration.146 0 .1% DDC diet cause chronic liver injury and induces very consistent and massive ov al cell accumulation after 4 to 6 weeks.147 In contrast t o the rat oval cell regimen, DDC do e s not completely block hepatocytes proli feration, 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 cell s share many similarities to rat oval cell s small in size (approximately 10m) and a large nucleus to cytoplasm ratio; they radiate from the periportal region forming primitive ductular structures wi th a poor ly defined lumen. 149 To isolate oval cell s from the liver, gradient density centrifugation is applied to separate nonparenchymal compartment ( NPC ) fraction contain ing oval cells from hepatocytes after two step collagenase perfusion of the liver.150 To further enrich oval cells from N PC, oval cell

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35 surface marker s 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, -glutamyl transpeptidase (GGT), OV 6 for rat and A6 for mou se ).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 Thy1+ oval cell s 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 u sing 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 the se protocols to induce oval cell s 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 hepati tis B158, and is also seen in the hepatocellular carcinoma and cholangiocarcinoma development.159 The Origin of Oval C ells The origin of oval cell s has been discussed for decades but still remains controversial. Traditionally, oval cell s have been believe d to originate in the liver within the C anals 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 der ived 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 Thy1, c kit, and CD34, are also expressed by oval cells. Additionally, sex -mismatched BM transplantation in lethally ir radiated DPPIV rat treated with 2 -AAF/CCl4 has firstly demonstrated cells in the bone marrow were capable of repopulating the

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36 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 cell s 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 hepa totoxic 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 Pathwa y of Oval C ell A ctivation The interaction between stromal derived factor 1 alpha (SDF a mediator of hematopoiesis165, has been proposed to be the possible mechanism by which oval cell s 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 receptor, CXCR4, while SDF protein expression was upregulated by hepatocytes in the injured liver. Oval cell s migrate along a SDF 1 chemotactic gradient to the injure d liver parenchyma. However, under nonoval cells aided regeneration, SDF 1 expression was not detected. SDF /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 mi togenic 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

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37 mitogenic effect on hepatocytes.167 Three primary growth factors, Hepatocyte growth factor (HGF), epidermal growth fac tor (EGF) and transforming growth factor are highly upregulated in liver regeneration via the stem cell compartment. HGF acts as a strong promoter of differentiat ion toward the hepatic lineage .168 Both oval cells and HSCs exp ress the HGF receptor c -Met .169 Transforming growth factor -beta1 (TGF alpha (TNF -ocytes and d own the myeloid lineage .170, 171 These cellular factors, plus yet undetermined factors, control the process of homing, engraf ting and differentitiating into a hepatic lineage. Bone Marrow C ells 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 tran s plantation gave rise to the 1990 Nobel P rize in Medicine Hematopoietic Stem C ells (HSC s ) Isolation and p henotype of HSCs HSCs are the best -studied and well -characterized population of stem cells mainly found in the bone marrow. HSCs can reconstitute the w hole 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 s hort 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 renew al and differentiation along any of the hematopoietic lineage.172 Strategies for preparing highly enriched

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38 HSCs may consist o f combination of lineage depletion followed by positive selection of Linsubset that express es 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 Linpopulation 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 a re sufficient to save 50% of lethally irradiated mice.173 HSCs in stem cell based gene therapy To date, appr oximately 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 disease s such as X -SCID, ADA SCID and CGD.176178 These clinical trails proved the concept of stem cell -based gene therapy but also point ed out potential barriers to the develop ment of th e 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, vir u s vectors that can transduce nondividing cells (e.g. HSCs) have been explored, such as lentiviral ve ctor and AAV. Another approach ha s been to stimulate HSCs to divide without differentiating by using cytokines, i.e., flt3 li ga nd and stem cell factor. Th e i nability to expand HSC s ex vivo hinders the current application of HSC s in both cell and cell -based gene therapy. This is especially true in case s where the number of available stem cells

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39 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 cel ls .162, 185, 186 These observations implies even broader application s of HSCs in both cell and stem cell -based gene therapy. Bone Marrow -D erived M esenchymal S tem C ells (BM -MSCs) MSCs are a heterogeneous population of plastic adherent, spindle -shaped and fibroblast like cells which can be extensively expanded in vitro and differentiate into mesenchymal lineage such as bone (osteoblasts), cartilage (chondrocytes), adipose (adipocytes). When cultured at low density, MSC s are able to form fibroblastic colonies, termed colony -for ming unit -fibroblasts (CFU -F). As another distinct stem cell population whic h resides in bone marrow, MSCs are a population of stem cell responsible for the maintenance of non -hematopoietic bone m a rrow elements which promote HSC proliferation and differentiation. The history of MSCs can be trace d back 130 years ago when the German pathologist Cohnheim first suggested the presence of non-hematopoietic stem cell s in his study of wound repair.187 It was not until the mid 1970s that Friedenstein and his colleagues first successfully isolate d fibroblast like cell s from bone marrow.188 Isolation and in vitro characteristics of BM -MSCs To date, Friedensteins 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 huma n, 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

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40 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 inve stigated to enrich mor e 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 speci es 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 Stro1 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 popu lation. Though not immortal, MSCs can expand in vitro many -fold and still retain the multi lineage potential. By culturing MSCs under conditions that are favorable for adipogenic, chondrogenic or osteogenic differentiation for 1 to 3 weeks, MSCs were highl y differentiated without evidence of the other lineages.189 In contrast, cultured primary fibroblasts, the mature mesenchymal cells, dont undergo any such differentiation under same induction condition. Differentiation capacity and immunosupression of BM -MSCs In addition to differ entiation 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 app eared to differentiate into car d i omyocytes after either site -directed or

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41 systemic delivery of MSCs. 193, 194 The t wo -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 gain ed cuboidal morphology of he patocytes and express ed liver specific marker gene in a time dependent manner. Differentiated cells further demonstrate d in vitro functional characteristic of liver cells including albumin production, glycogen storage, urea secretion, uptake of low -density lipoprotein and cytochrome P450 activity.198 Hepatic preconditioned human MSCs functionally integrated into hepatectomized mouse liver administered by intrasplenic injection.195 A n important advantage of using M SCs is that in some situatio n they dont need to be matched, since the immune phenotype of MSCs (widely described as MHC I+, MHC I I ,CD40 ,CD80 and CD86 ) is considere d as nonimmunogenic ; therefore transplantation into an allogeneic host may not induce immunoresponse .190 This provides the pos sibility 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.199201 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 C ell F usion Transdifferentiation, the conversion of adult stem cells from one l ineage to another, has been proposed to explain the observation that transplanted bone marrow stem cells can turn i nto unexpected lineages such as myocytes, endothelial cells, hepatocytes, neurons and many others 145, 203205 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

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42 therapy. In order to identify the molecules that are respon sible 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 respecti vely 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 cel ls206, 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 transdiffe rentiation. 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 tra nsformation 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 mean s 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 T issue -D erived Mesenchymal Stem C ell s (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 tissu0065.209212 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

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43 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, t he colony frequency is hi gher i n adipose tissue than in bone marrow e.g. the number of CFU -F was 557673 for adipose tissue vs 8361 for bone marrow at an initial plating density of 1106 cells per cm2, as well as the maintenance of proliferating ability in culture.197, 215, 216 H arvest ing of BM is a lso a highly invasive procedure and the number, differentiation potential, and maximum life span of MSCs from BM decline with increasing age 217219. 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 C haracterization of AT-MSCs Adipose tissue, li ke 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 researche r s have found the liposuction waste are an alternative source of adult stem cells that are believed to contribute to repair and healing as regenerative med icine 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. E ach adipose tissue type serves a distinct bio logical 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

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44 Rodbell and Jones presented the first in vitro isolation method for mature a dipocytes 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 cir culation 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 3 08,849 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 name s h ave 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 (AD MSCs), and processed lipoaspir a te (PLA) cells, etc. The surface immunophenotype of hu man AT -MSCs resembles that of human BM derived MSCs, with > 90% identity based on direct comparison221, that is nega tive to endothelial marker ( CD144) or HSC maker ( CD34, CD45, CD 14) but positive to typical MSC marker (CD29,CD44,CD73, CD90,and CD105).197 Discrepanc y has been observ ed 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 on ly on 0.8%54% of the initial SVF, and on up to 98% of AT -MSCs at late passage.222 P roliferation and Differentiation C apacity of AT -MSCs Following isolation, human AT -MSCs remain inactive with an initial lag ti me of 5 7 days, and then enter a proliferative phase, reaching confluence within 48hr s .210 Human AT -MSCs

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45 display a cell doubling time of 2 to 4 days depending on culture medium and passage number222, 223 or 60h rs 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 expa nded routinely beyond 180210 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 k aryotype and tumor formation when implanted into immunodeficient mice, which might be due to the overexpression of oncogene c -my c 225 Not only can AT -MSCs differentiate into mesodermal lineages e.g., adipogenic, o steogenic, 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, 226231 Evidenc e 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 function s such as albumin production, low density lipoprotein uptake, and ammonia detoxification, aft er 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 ly mphoid 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 lineag e -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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46 behind the cross -differentiat ion is far from clear and more research is needed to deco de this puzzle for future AT -MSC -related cell therapy and tissue engineering. For clinical purposes, adipose ti ssue 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 A natomy 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 nutrie nts 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 ce lls 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 drain s 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 sinuso id), stellate cells (located under the sinusoid), vascular endothelial cells, and pit cells (natural killer cells). Among these, hepatocytes are responsible for the majority of li ver function

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47 and constitute ~60% of the liver cell population and 90% of li ver mass. An adult mouse liver contain s about 5107 hepatocytes and an adult human liver contains about 8 010 hepatocytes.235 Hepatocytes are the largest and polygonal -shaped epithelial cells ( 3040m) with diploid or tetraploid nuclei and a large proportion of adult hepatocytes are binucleated.236 Zone 1, 2 and 3 hepatocytes are distinguished based on the basis of their relative position within the lobule. Zone 1 hepat ocytes 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 nu cleated 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 s erum 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 hr s 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 st arts 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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48 CHAPTER 2 MATERIALS AND METHOD S 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 nonparench ymal (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 usi ng Sca I antibody conjugated magnetic beads (Miltenyi Biotec, Germany) Bone Marrow I sola tion 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 n eed le 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 70m nylon mesh strainer (Becton Dickinson Labware, Franklin Lakes, NJ). C ells were treated with red blood cell (RBC) lysing buffer for 2 minutes at room temperature to deplete RBC. After that c ells were cultured for 1h at 37 to remove the macrophages which will attach to the bottom of the c ell culture dish. Those remaining suspension cells contained mesenchymal and hematopoietic stem cells and blood progenitor cells and were ready for virus ve ctor transduction. Transduction was performed in a 1ml reaction

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49 volume of DMEM supplemented with 10% FBS for 2 h at 37 and 5% CO2. Cells were the washed three times with PBS and resuspend ed in 100ul saline for transplantation or cultured in murine myeloid longterm culture medium (MyeloCultTM M5300, StemCell Technologies Inc., Vancouver, BC) for in vitro transduction efficiency study. AT -MSCs I solation 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 37C with gentle agitation. The collagenase was inactivated with an equal volume of DMEM (Media tech, Inc., Manassas, VA) supplemented with 10% fetal bovine serum (FBS, HyClone laboratories, Inc., Logan, Utah), and the infranatant was centr i fuged at 1,000 rpm for 5 min at room temperature. The cell pellet was resuspended in 160mM NH4Cl (StemCell Technologies Inc, Vancouver,BC) and incubated at room temperature for 2 min to eliminate contaminating red blood cells and filtered through a 100m nylon mesh strainer (Becton Dickinson Labware, Franklin Lakes, NJ) to remove debris. The resulting AT -MSC -cont aining cell pellet is collected by centrifugation as described above, and resuspended in a DMEM/10%FBS medium at 1 2106 cells/100 mm plastic tissue culture dishes. Nonadherent cell population was poured off after 12 16hr culture. Adherent cells were wash ed with PBS and cultured in DMEM with 10% FBS for expansion. The cell s with 70 80% confluence were harvested with 0.25% t rypsin-EDTA and reseeded at 1.0 105 cells/60mm dish. Recombinant AAV V ector 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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50 cDNA flanked by two ITRs and driven by CMV enhancer/ chicken -Actin promoter, i ntron 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 D sequence 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 w as replaced by duck hepatitis B virus (DHBV) promoter (Figure 2 1 B) or CMV promoter (Figure 2 1 C). Plasmid ssAAVCB -hAAT, dsAAVDHBVhAAT, and dsAAVCMV -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 gene s from adenovirus and corresponding serotype AAV cap and rep genes into 293 cells. Cells were harvested and disrupted by freeze -thaw lysis to release virio ns 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 pTYF linker vector which was derived from an LTR -modifi ed recombinant HIV 1 plasmid This l entiviral vector was packaged by as previously described. Briefly pHP -helper (packaging helper construct), pHEF -VSV G (envelop e xpression 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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51 Amicon Ultra 15 centrifugal filter device (Millipore) The virus titer was estimated at 1109 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 1104cells/well and infected with virus vectors at different multiplicities of infection (MOI) in triplicate. Oval cells were cultured in Iscoves Modified Dulbeccos m edium (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 Injec tion of V ectors into Mouse Liver and M uscle For muscle injection, 8 -week old female C57BL/6 mice were anesthetized by i soflurane inhalation, and aliquots of vectors were injected percutaneously into the quadriceps femoris muscles of both hind limbs .243 For p ortal vein inject ion, mice were anesthetized by i soflurane inhalation and a ventral midline abdominal incision was made into the peritoneal c avity 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 21010

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52 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 acidifi ed 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 o f 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 f urther studies were conducted. Adipogenic and Osteogenic D ifferentiation of AT-MSCs Adipogenesis At pas sage 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 sup plemented with 10% FBS, 0.5mM 3 isobutyl 1 -methylxanthine, 1M dexamethasone, 200M indomethacin, and 10 g/ml bovine insulin (all Sigma). Adipogensis was assessed by staining for intracellular lipid droplets with Oil Red O stain (Sigma). Osteogen e sis Cultured c ells 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 consist s of glycerophosphate, and

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53 50 M ascorbate 2 phosphate (all Sigma). Osteogenesis was assessed by staining for calcium depositions with Alizarin Red S (pH 4) stain (Sigma). Liver Directed Transplan tation of A dult Stem C ells Adult fema le 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 hepatectomiz ed 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 immediate ly 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 s ite 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. Immuno histochemistry for Human AAT, GFP and Mouse Albumin Organ tissues were fixed in 10% neut ral buffered formalin (NBF) and embedded in paraffin. For hAAT and GFP immunostaining, tissue secti -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,

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54 Carlsbad, CA, USA) for 5 min utes 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 minut es 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 b y Vectastain ABC Alkaline Phosphatase kit (Vector Laboratories) Vulcan Fast Red (VFR) chromagen (Biocare Medical, Concord, CA, USA). I mmunofluores c ence 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 a ntibody were applied Immunofluorescent S taining 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 gly cine 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 mon oclonal 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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55 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 Fluores c ence in situ Hybridization sections of paraffin embedded liver tissue samples were used for detecting Y chromosome. Liver sections were de paraffin ized 25 min in fresh xylene and rehydrated 22 min in 100% ethanol 2min in 95% ethanol, 1 min in 70% ethanol and 1 min in water. De paraffinized 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 2SSC and then dehydrated through graded alcohols. Sections were incubated with FITC -conjugated Y chromosome 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/2SSC, 2SSC, and 4SSC+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 DAP I (Vector, Burlingame, CA). Human AAT S pecific ELISA Microtiter plates were coated with 100ul of goat anti hAAT (1:200, Sigma Imm unochemical, St.Louis, MI, USA) in vollers buffer overnight at 4oC. Blocking buffer, 3% bovine serum albumin (BSA Sigma, St.Louis,MI,USA), wa s 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 incubate d 1hr at 37oC. A second antibody, r abbit anti -hAAT (1:1000, Roche Molecular Biochemicals, Indianapoli s,IN, USA), was added and

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

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57 A ITR CB promoter h AAT pA ITR B Delt ITR DHBV promoter h AAT pA ITR C Delt ITR CMV promoter h AAT pA ITR Figure 2 1. rAAV vectors constructs ( A) rAAV-CB -hAAT. P lasmid rAAV-CB hAAT contained full length AA V2 ITRs, hAAT cDNA driven by CMV enhancer/ chicken -Actin promoter, intron and ploy(A) sequence. (B) rAAVDHBV-hAAT. D -sequence and terminal resolution site (trs) of 5 -ITR i s deleted to make self -complementary dsAAV vector. 3 ITR remains intact. hAAT is driven by DHBV promoter. (C) rAAVCMV hAAT. 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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58 CHAPTER 3 HEPATIC OVAL CELL -BASED L I V ER GENE D ELIVERY Introduction Our previous study has shown that hepatic oval cell s 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 T he goal of this study is to further evaluate the potential of viral vector -mediate d adult stem cell ba sed therapy. Since transgene expression is affected by transduction efficiency of viral vector, transplantation efficiency, and engraftment capability of adult stem cell s, we have tested the possibility of enhancing oval cell transduction efficiency by optimizing rAAV vectors and employing a lentiviral vector Animal Experimental Design Fres hly isolated liver oval cells ( 2 106 cells) from male C57BL/6 mice were infected wi th rAAV1 CBhAAT 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 intrasple nic 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 week s to stimulate oval cell proliferation. Recipient mice were injected with monocrotaline (MCT) 50mg/kg BW twice at a 2 -week interval to inhibit the endo genous liver cell proliferation. Two weeks after the second injection, recipient mice were partially hepatectomized to remove 70% of t he liver to create liver injury and thus enhance the environment for the proliferation of transplanted oval cells (Figure 3 -

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59 1 ). A spe cies -specific hAAT ELISA was performe d to measure serum hAAT protein level from the transgene expression. At the end of experiment, liver tissue s were collected for immunostaining for hAAT. Results Ex vivo Transduction Efficiency on Oval Cells b y rAAV Vector s 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 pri mates or bovine rAAV vector using GFP as a reporter gene, respectiv ely. An identical genome, AAV CB GFP, were packaged into each of four different AAV serotype capsids (serotype 1, 7, 8, 9) and bovine AAV capsid. rAAVCBGFP vector transduced oval cells were subjected to flow cytometry analysis to quantify the GFP positive o val cells. As seen in Figure 3 2 rAAV1 -CB GFP vector yielded 2.17% green fluorescent cells 7 day after infection at a dose of 104vg/cell. rAAV7 CBGFP and rAAV8 CB GFP vectors yield ed 2.11% and 1.63% green fluorescent cells, respectively, under the sam e condition. rAAV9 and b ovine 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, althou gh the transduction efficiency wa s low. Lentiviral Vector Construction In order to generate a lentiviral vector expressing hAAT, a lentiviral virus vector plasmid, pTY F linker derived from HIV 1 was used as a parental plasmid (Figure 3 3A). Human AAT expression cassette including hAAT cDNA drive n 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. S ince 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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60 cDNA We have inserted an enzyme digestion site SpeI in to th e 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 a mplified fragment w as cloned into TA clone and released by enzyme digestion with BstEII and SpeI. The BglII BstEII (2639 bp) fragment and the PCR amplified B stEII -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 b y Dr. Changs laboratory. To evaluate the transduction efficiency of Lenti -CB -hAAT vector on oval cells, oval cells were infected with Lenti CBhAAT vector at 5 MOI (multiplicity of infection) To compare the transduction efficiency between lentiviral vect or 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 C ells Based on above results we chose rAAV1 CB -hAAT and Lenti -CB -hAAT vector s for transplantation studies. In this study, each recipient mouse received rAAV1 -CB hAAT or Lenti CB hAAT vector transduced oval cells ( 2106 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

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61 with rAAV1 CB -hAAT vector transduced oval cells exhibite d transient elevation (2,500ng/ml) of serum hAAT at week 2 post transplantation. Other mice in both Lenti CB -hAAT vector and rAAV 1 CBhAAT vector groups didnt 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 modif ication 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 b y 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. Hos t 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 bet ter 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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62 oval cells among different AAV serotypes (serotype 1 5 and 7 9), and bovine AAV. Hence rAAV1 CBhAAT vectors were used for in vivo transplantation study. We observed transgene expression in the recipient liver after transplanting rAAV1 CB hAAT vector transduced oval cells. We also noticed the large variation amo ng 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 demo nstrated 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 c irculating 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 tha t Lenti -CB -hAAT vector infected oval cells efficiently and mediated efficient transgene expression in cell c ulture 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 administrat ion 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 additio n, 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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63 provirus and trans -factor s 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, 253255 Although our studies have clearly demonstrate the feasibility of stem cell mediated hAAT gene delivery using rAAV vector and oval cells, i solation of oval cell for autologous transplantation in human is not practi cal. Therefore, next studies will focus on finding new stem cell resources for liver directed hAAT gene delivery.

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64 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 tra nsplantation. 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 i nto the recip ient liver by intra s pl e nic injection. Serum hAAT levels were monitor by ELISA. Liver tissues were subjected to hAAT specific immunostaining.

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

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66 Figure 3 3. Const ruct 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 -hAA T plasmid ( D) Gel electrophoresis results. H: HindIII; SB: SpeI & BamHI; S: SpeI; B: BamHI.

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67 Figure 3 4 Ex vivo transduction of oval cells. Mouse oval cells wer e grown in the 24 -well plate (1 104 cells/well; n=3) and were infected with the Lenti -CB -hA AT vector at 5 MOI rAAV1 CBhAAT 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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68 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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69 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 2h r, respectively, and transplanted into hepatectomized m ouse 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 transie nt increase in serum hAAT level at week 2 post transplantation. Dash is lower limit of quantification (LLOQ).

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70 CHAPTER 4 EX VIVO TRANSDUCTION AND TRA NSPLANTATION 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 f or liver gene delivery of human alpha 1 antitrypsin (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 v itro studies showed that bone marrow cells can be transduced by lentiviral vector (Lenti -CB -hAAT) and recombinant adenoassociated viral vectors (rAAV1 CB hAAT, and rAAV8 CB -hAAT). Transplantation studies showed that transplanted bone marrow cells homed in to liver, differentiated into hepatocytes and expressed hAAT in the liver. Importantly, we showed that transplantation of rAAV8 CBhAAT vector transduced bon e marrow cells resulted in long term and sustained levels of hAAT in the systemic circulation of re cipient 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 secr eted into the circulation and consequently leads to a reduced level of AAT in the serum.257 As a serine protease inhibitor, t he primary function of AAT is to protect delicate tissue such as pulmonary interstitial elastin against the excessive

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71 proteolytic damage of neutrophil elastase (NE) Deficiency of AAT in the serum could cause alveolar e lastin 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 11M (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 patien ts 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 wildtype AAT gene to correct the deficiency of AAT by gene transfer to muscle are being investigated in the phase I clinical tria l using recombinant adenoassociated virus vector ( r AAV) 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 do nor organ and immune rejection. Adult stem cells offer a platform for ex vivo genetic manipulation followed by autologous transplantation, which will overcome many limitati ons, 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 s tem 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 ge ne therapy may reduce or eliminate the need for repeated administration of the gene therapy. A dult stem cell gene therapy which replaces the patients disease -causing gene with the healthy

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72 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 progenit or cells .145, 162, 164 Sex mismatched B M 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 propos ed 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 differenti ation, longterm persistence an d 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 appl ied for gene targeting in MSCs.110 In the present study we tested t he 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 an d received PHx before transplantatio n. 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 infe cted with L enti -CB -hAAT vector at 1102 MOI, r AAV1 -CB hAAT and

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73 rAAV8 CBhAAT vector at 1104 MOI, respectively, for 2 hours. After transduction, BM cells were washed 3 times with PBS and then resuspended in saline solution at an ap proximate concentration of 5 06 cells/100ul. Transduced BM cells were transplanted into the recipient liver (5 06 cells/mouse) by intraspl enic injection (n=4) or portal vein injection (n=5) Serum samples were collected every week post transplantation. Serum hAAT levels were mon itor ed 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 C ells Transduction In order to test the transduction efficiency of bone marrow cells, total bone marrow cells were isolated and infected with Lenti CBhAAT, rAAV1 CB -hAAT and rAAV8 CB -hAAT vectors. As shown in Figure 4 2 all vectors can transduce BM cells, but with differen t 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 CBhAAT 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 T ran splantation of ex vivo Transduced Bone Marrow C ells Next, w e 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 wer e 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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74 the environment for the proliferation and differentiatio n of transplanted bone marrow cells. In this experiment, freshly isolated total bone marrow cells (donor cells) were infected with Lenti CB hAAT (MOI=100), rAAV1 CB -hAAT (MOI=1x104) and rAAV8 CB -hAAT (MOI=1x104) vectors, respec tively. After thorough washes 5 x106 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 CBhAAT vector treatment group were also GFP positive Bone Marrow Cell Transplantati on 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 modifie d procedure .245 In this experiment, we transplanted 2x107 rAAV8 CBhAAT infected bone marrow cell s ( from male C57BL/6) into the recipients through intra splenic 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 mos t 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 ( Fig ure 4 7 A and 4 7 B). AAT -immunostaining also showed some AAT -positive cells in lung and bone marrow indicating that intra splenic 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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75 arrow). Important ly, longterm 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 represent s a novel therapy for AAT deficiency Discussion Adult stem cells offer a platform for ex vivo genetic manipulation followed by autologous transplantation. Ad ult 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 patients stem cells carrying healthy gene(s). We have pre viously 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 investigate d 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 t herapy 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, tra nsplantation of these transduced cells resulted in undetectable levels of hAAT in the circulation, although some hAAT positive cells were detected in the recipien t 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 c ells 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 CB hAAT infected BM cells than that from recipients received rAAV1 CB -hAAT infected BM

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76 cell s. 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 tha t 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 hepatic differentiation potential.162, 195, 198 Furthermore, considering the possible contribution of cell to cell interaction to stem cell proli feration and differentiation the present study employed total bone marrow cell s 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 trapp ed in the injection site, spleen, while some cells home back to the bone. The BM migrat ing 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 hepatocyt es 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 improve ment on transgene expression in the donor cells is required to achieve therapeutical applicat ion 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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77 establish long -term and persistent gene expression. Integration to AAVS1 locus could resist to transgene silencing, a major obstac le 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 vect or 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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78 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 CBhAAT, rAAV1 CB -hAAT rAAV8 CB hAAT vector respectively, for 2 hours, washed and transplanted into the recipient liver by portal vein injection or intra splenic injec tion. Serum hAAT levels were monitor by human AAT specific ELISA. Liver repopulation was measured by immunostaining.

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79 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, Lenti CB 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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80 Figure 4 3. Detection of expression of human alpha1 antitrypsin (hAAT) in recipient liver aft er transplantation of viral vector infected BM cells by immunostai ning (A) Liver section from C57B L/6 mouse transplanted with Lenti CB -h AAT infected BM cells (Brown). (B) Image A view at larger magnification. (C) Liver section from C57B L/6 mouse transplanted with rAA1 CBhAAT infected BM cells. (D) Image C view at la rge r magnification. (E) Liver section from C57B L/6 mouse transplanted with rA AV8 CBhAAT infected BM cells. (F) Image E view at larger magnification. (G ) Human liver sect ion served as positive control. (H) Liver section from untransplanted C57BL/6 mouse serve d as negative control.

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81 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 tr ansplanted with rAAV8 CB hAAT 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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82 Figure 4 5 Detection of donor ce lls 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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83 Figure 4 6. Detection of coexp ression of human alpha 1 antitypsin (hAAT) and mouse albumin by immunostaining. (A C ) Liver section from C57BL/6 mouse transplanted with rAAV8 CBhAAT infected BM cells stained for hAAT (brown). (B,D) Liver section adjacent to the section in A and C, resp ectively, 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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84 Figure 4 7. Multi organ homing of transplanted BM cells. Tis sue sections were from female C57BL/6 mouse at 8 weeks after transplantation with rAAV8 CBGFP vector infected male BM cells. (A) S pleen section subjected to FISH for detecting Y chromosome. (B) Spleen section stained for GFP (brown). (C) Bone section stai ned for GFP. (D) Lung section stained for GFP. Black arrowheads indicate the observed GFP staining.

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85 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 1104 particle s/cells for 2 h and transplanted into liver of partially hepa tectomized C57BL/6 recipient ( 2 x107 cells/mouse; n=4 ) by intrasple nic 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). T he serum level of hAAT from untransplanted C57BL/6 mouse (negative control) was below the LLOQ.

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86 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 mesen chymal 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 differentiat ion of AT -MSCs, our study investigated the feasibility of AT MSC -based liver gene delivery to correct a genetic disease, alpha 1 antitryspin deficiency (AATD). In vitro study showed AT -MSCs can be efficiently transduc ed by recombinant adeno associated vira l vector serotype 1 (rAAV 1 -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 se rum hAAT level of 100 200ng/ml Immunostaining for hAAT on recipient liver section revealed that about 5 10% recipie nt liver was repopulated from approximately 1.6 06 ex vivo genetically modified AT MSCs, 8 week s post transplantation. More importantly A T -MSC derived 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, contribute d to liver regeneration, and serv ed 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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87 Introduction Alpha 1 antitrypsin (AAT) deficiency is a genetic disorder resulting from a sing le gene mutation on AAT coding gene, which results in a red uc tion of serum levels of AAT and accumulation of AAT protein in hepatocyte s C onsequently this mutation causes an increased risk of developing early onset pulmonary emphysema and severe forms of l iver 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 availab le treatment for AAT def iciency 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 o f 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 hepatocyte s for l iver 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 fibroblast like cells which can be extensively expan ded 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 viv o .195, 198 MSCs have been traditionally isolated from bone marrow aspirates with a yield of approximately 1 MSC per 105 BM nucleate d 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 B M decline with increasing age. 217219 Fortunately, MSCs can also be isolated from adipose tissue.221, 261 There is little to no

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88 difference between BM MSCs and AT -MSCs regard ing 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 c olony -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. Adenoassociated virus (AAV) is a linear single -stranded DNA parvovirus with a genome of 4.7 kb and a non-enveloped capsid of app roximately 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 b e 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 adm inistrated to a human subject f or 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 deve loping new AAV vector by modifying both AAV genome and capsid protein. Fo r 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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89 synthe sis of dsDNA from the ssDNA .86 Other efforts have focused on engineering capsid protein such as transcapsidation, adsorption modification mosaic capsid, and chime ric 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 hav e shown hundred-fold more potency for murine musc le tran sduction 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 longt erm 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 b e 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 F emale C57BL/6 mice (8 -week old) were in jected 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 percutane ous injection into the quadriceps femoris mu scles 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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90 every week post injection and subjected to hAAT specific ELISA to evaluate the transgene, hAAT, expression. Ex vivo Transduction and T ransplantation of AT -MSCs The recipients, 4 -week old female C57BL/6, were IP injected twice (2 weeks interval) with MCT at 50mg/kg BW an d received PHx before transplantatio n. AT MSCs were isolated from the peritoneal adipose tissue of 6 to 8 -week old male C57BL/6 mice. The newly isolated AT MSCs were infected with ssAAV1 CB -hAAT vector at 5104 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 1.606 cells/100ul. Transduced AT -MSCs were transplanted into the recipient liver (1.6 06 cells/mouse, n=5) by intraspl enic injection. Serum samples were collected every week post transplantation. Serum hAAT levels were monitor ed by ELISA. 8 weeks post transplantation, liver tissue of recipient mice were harvested for immunostai ning (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 i mmunofluorescence and multiple differentiatio n 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 didnt express eithe r hematopoietic markers CD34 and CD45 or endothelial marker CD31. The expression of CD133 was low ( Figure 5 2A). The c ell surface phenotype s were consistent with those reported in the literature for adipose tissue derived stem cells.222 Multiple differentiation potential of AT -MSCs was dem onstrate d by induced differentiation in to adipocytes and osteocyte s. Two week s after

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91 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 V ecotors AAV is a single stranded DNA virus. After infection, the viral DNA undergoes second stranded DNA synthesis usi ng host cellular enzymes. Therefore, transgene expression from conventional single stranded rA AV vector depends on the second-stranded DNA synthesis. Recentl y double -stranded AAV vectors ( d s AAV), or self -comp lementary 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 ITR s It has been shown that dsAAV can avoid second stranded synthesis in the host cells thus mediated quicker and high er levels of transgen e expression. In order to achieve optimal levels of hAAT expression in AT MSCs, we have generated two dsAAV vectors, dsAAVDHBVhAAT 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 w as 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, dsAAV 1 CMV -hAAT mediated detectable levels of hAAT expression. H owever, the levels were lo wer than that from matched dose of ssAAV 1 CB -hAAT vector. Similarly, the dsAAV DHBVhAAT vector plasmid was packaged into rAAV8 vector for liver gene delivery. As shown in Figure 5 3B, dsAAV 8 DHBVhAAT mediated sustained levels of hAAT, but the levels were much lower than that from ssAAV 8 CB -hAAT vector These results suggest ed that advantage of

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92 dsAAV vector in muscle and liver was limited and did not overcome the advantage of CB promoter. In order to select the most efficient rAAV vectors for AT MSCs matched dose of all above vector s were used to infect AT -MSCs. As shown in Figure 54A, ssAAV1 CB -hAAT vector mediated the highest transgene expression than the other three r AAV vectors, dsAAV1 CMV -hAAT, ssAAV8 CB -hAAT, and dsAAV8 DH BV hAAT, as indicated by more than 10 fold increase in hAAT serum level at day 9 post transduction. It wa s interesting that rAAV 1 mediated more than 25-fold higher level s 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 transductio n 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 CBhAAT could serve as a platform f or liver ex pression of hAAT after autologous transplantation. To test this hypothesis, AT MSCs (1.6 106) from male mice were infected with ss AAV1 CB -hAAT (MOI=5x104) and were t ransplanted into the liver of MCT -treated and partial -hepatectomized female C57 BL/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 subsequentl y 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

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93 immunostaining. Immunostain in g for hAAT revealed that 5 10% of total hepatocytes displayed hAA T transgene expression (Figure 5 5 ). Those hAAT positive hepatocytes indicated that rAAVtransduced AT -MSCs were able to migrate into liver from the injection site of spleen, engraft in to the recipient liver parenchyma contribute to liver repopulation, and give rise to transgene expression. More importantly, those AAT positive cells were morphologically similar to hepato cyte. Y -chromos ome f luorescent 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, respectivel y. 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 G FP (Figure 5 8). In order to quantify transgene product generated and secreted from engrafted rAAV transduced 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 d emonstrated 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 MSC s for liver regeneration and gene delivery remain elusive. In this study, we isolated and characterized mouse AT -MSC s We showed that these AT MSCs ca n be efficiently transduced by ss AAV1 CBhAAT vector. Tr ansplantation of rAAV transduced AT -MSC s

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94 resulted in hepatic different iation and sustained long term transgene expression in the liver an d the serum of the recipients. Results from this study demonstrated that it is feasible to use AT MSCs as a cell vehicl e for li ver 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 11M or 800g/ml may develop emphysema. Therefore, the se rum level of hAAT obtained from AT -MSC s transplantation in this study remain further im pr ovement to be therapeutic (approximately 500800ug/ml)12. Several possible approaches may be employed to e nhance 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 MSC s 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 a nd collect putative multipotent stem cell from mouse adipose tissue -derived stromal vascular fraction culture cells (ADSVF cells).265 Secondly, hepatic -differentiation potential of AT MSC s can be further improved by in vitro preconditioning toward h epatocyte like cells such as incubating MSCs with specific growth factors e.g., hepatocyte growth factor (HGF), epidermal growth factor (EGF) or fibrobl ast growth factor (FGF).226, 232 In addition, improvement of tr ansduction efficiency of AT -MSC by further optimize r AAV 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 vector s and recently developed mutant AAV vector s might mediate higher transduction efficiency in AT -MSCs. In contrast to previous study 87, our s tudy showed dsAAV vectors didn t

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95 demonstrate superior transduction efficiency to ssAAV vector. This inconsistency to the well documented characteristics of dsAAV might reflect the inferior promoter activity of DHBV and CMV, compared to the CB promoter. Pre vious studies have shown a 10100 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. Conside ring the dilution of episomal r AAV vector during cell division, site -specific integration AAV vector system may not only enhance but also ensure a longterm transgene expression. Importantly, AAVS1 site has been reported to be a safe integration site.46 A bipartite rAAV vector has been designed to fulf ill this concept.101 In addition to the feature of easy isolat ion in large quantity from adipose tissue and expendable in vitro AT -MSCs, like bone -marrow derived MSCs, also exhibit immunomodulatory and anti prolife rative 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 re cently -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 o ur study using rAAV vector expressing hAAT gene consisted and further extend ed the previous observations232 thus paved a path to both basic and clinical studies.

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96 Figure 5 1. Experimental outline of AT MSCs study The recipients ( female C57BL/6) were IP injected twice (2 weeks in terval) 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 CBrAAV-CB -hAAT vector for 2 hours, washed three times with PBS, and transplanted into the recipient liver by intra splenic injection. Serum hAAT levels were monitor by human AAT specific ELISA. Liver repopulation was measured by immunostaining.

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97 A. B. Figure 5 2 Characterization of AT MSCs. (A) Expression of cell surface markers in AT MSCs by i mmunofluorescence staining (B) Multip le differentiation potential of AT MSCs. Left, undifferentiated AT MSCs; Middle, AT -MSCs were cultured for 2 week s 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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98 A B Figure 5 3 In vivo mu scle 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 -hA AT 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 CBhAAT vector; O pen square dsAAV8 DHBV hAAT vector; Solid triangle, saline group s erve as negative control.

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99 A. B. 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 (5104cells/well, n=3) a nd infecte d with rAAV hAAT 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, ssAAVCBhAA T vector; Cross, dsAAVD HBV-hAAT vector; Dash, lower limit of quantification (LLOQ). hAAT level of PBS group ( nega tive control) was below LLOQ. (B) D ouble transduction of AT MSCs by ssAAV1 CB -hAAT vector Mouse AT -MSCs (passage=1) were seeded in 24 -well (1 104cells/well, n=3) a nd 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, t wo infections at 12 hr interval; Dash, lower limit of quantification (LLOQ). hAAT leve l of PBS group ( negative control) was below LLOQ.

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100 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 C57B L/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 a s positive control (F ) Liver section from an untransplanted C57BL/6 mouse serves as negative control.

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101 Figure 5 6 Detection of donor cells in recipient liver after AT -MSCs transplantation by fluorescence in situ hybridizations for Y -chromosome (A) Ma le 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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102 Figure 5 7 Detection of coexpression of human alpha 1 antitypsin (hAAT) and mouse albumin by immunostaining.( A) Liver section from mouse t ransplanted with ssAAV1 CB hAAT 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 contr ol. (D) Normal mouse liver section staining for mouse albumin served as positive control.

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103 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 CBGFP vector in fected 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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104 Figure 5 9. Detection of e xpression of human alpha 1 antitrypsin (hAAT) in the serum. AT MSCs from C57B L /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). T he serum level of hAAT from untransplanted C57BL/6 mouse (negative control) was below the LLOQ.

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105 CHAPTER 6 SUMMARY AND FUTURE DIRECTION Summary Protein replacement therapy has been only optional treatment for AAT deficiency for more than 20 year s 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 th e use of r AAV 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 live s of people suffering from leukemia lymphoma and other inherite d blood disorder s. 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 an d differentiation potential of stem cells. In 1990, the first gene therapy trial was a cell based gene therapy. Patients T -cells were isolated and transduced with MoMLV -ADA vector ex vivo followed by return ing to patients. Patients benefited and suff ered no harmful effect from this therapy, however, s ince 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 perman ent 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 a nd 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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106 term transgene expression by integrating viral genome into host chromosome. Both viral vector s represent unique property in the gene therapy. To answer the question that whether genetically modified adult stem cells can be utilized to correct the gen etic 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 adu lt 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 transpla nted BM cells into mouse liver s 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 allogr aft transplantation. These results showed that BM cells can be transduced by rAAV and lentiviral vectors and engrafted into liver re sulting 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 represent s an ideal source of autologous stem cells, AT -MSCs. Liposuction results in minimal patient discomfort and adi pose 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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107 transduced an d 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 s howed adult stem cell s 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 o f 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 o f adult stem cell based gene therapy is determined by three main factors, transduction efficiency, engraftmen t efficiency, and transgene expression. Optimization in any of these three factors will definitely contribute to achieve our goal For increasing t ransduction 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 vector s 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 expressio n. Site -specific integration provides a long -term treatment and reduces the risk of tumor i genesis 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 s tem cell homing and engraftment are a complex and multistep process. These evolve v arious adhesion receptor s and ligand s that mediate cell to -matrix and cell -to -cell interaction, including selectins, integrins, and Ig family and numbers of others undefined factors.

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108 Several signaling pathways (e.g.SDF1 researches are needed to solve this puzzle. Tumor formation is a big issue for stem cell therapy. Detail examination of the tumorig enesis of stem cell is required to make stem cell an effective and safe therapy.

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130 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 S chool in 1997, she started her college education at China Pharmaceutical University in Nanjing P. R. China She was awarded her b achelor s degree in b iotechn ical p harmaceutics in 2001 and m aster s degree in microbiology and biochemical p harmacy in 2004. S he 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 m asters degree in s tatistics in 2008 from University of Florida, Department of Statistics.