adult stem cell-based gene therapy for alpha 1...
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ADULT STEM CELL-BASED GENE THERAPY FOR ALPHA 1-ANTITRYPSIN DEFICIENCY
By
HONG LI
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2009
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© 2009 Hong Li
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To my parents, Guoji Li and Baozhu Zheng, and my sister, Xia Li
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ACKNOWLEDGMENTS
I would like to express my deepest gratitude to my mentor, Dr. Sihong Song, for giving me
the opportunity to pursue my Ph.D. degree in his laboratory. Dr. Song was always there to help
me, listen to me and offer advice. He showed me different ways to approach research problems
and guided me development as a scientist. His enthusiasm for science continues to inspire me to
overcome the difficulties during my research.
I would also like to thank the members of my dissertation committee: Dr. Guenther
Hochhaus, Dr. Jeffrey Hughes, and Dr. Bryon Petersen; for their valuable suggestion, and
advice.
Furthermore, this project would not have been possible without the kind help of Dr.
Young-Kook Choi and Dr. Bin Zhang, who both taught me molecular cloning techniques; Dr.
Yuanqin Lu, who assisted with my animal studies; Dr. Rafal Witek, who taught me how to do
liver transplantations; and Marda Jorgensen, for teaching me Y- FISH. I would also like to
acknowledge Dr. Martha Campbell-Thompson and the staff of the Pathology Core; especially,
Amy Wright and Dontao Fu, for their assistance with immunohistochemistry assays. Also many
thanks go out to all the members of Dr.Song’s lab, in particular, Dr.Christian Grimstein and
Matthias Fueth, who supported me in all my endeavors.
Last, but not the least, I would like to thank my parents, Guoji Li and Baozhu Zheng for
their unconditional support and guidance in my life. Without their tremendous support, I would
not have been able to realize this dream. I also extend a special thanks to my sister, Xia Li. She
encouraged me to study abroad and face the challenge. She is always by my side, listens
patiently to my frustrations, and helps me through the tough times. I also thank my 3-yr-old
niece, Minghui Zhang, for her to bring happiness to my life.
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS.................................................................................................................... 4
LIST OF FIGURES .............................................................................................................................. 8
ABSTRACT ........................................................................................................................................ 10
CHAPTER
1 LITERATURE REVIEW ........................................................................................................... 12
Introduction ................................................................................................................................. 12 Alpha 1-Antitrypsin Deficiency ................................................................................................. 12
AAT Biology........................................................................................................................ 13 AAT Deficiency Pathogenesis ............................................................................................ 14 Current Therapy for AAT Deficiency ................................................................................ 16
Recombinant AAV Vector ......................................................................................................... 19 AAV Genome ...................................................................................................................... 19 AAV Entry ........................................................................................................................... 20 AAV Serotype ...................................................................................................................... 22 Self-Complementary AAV.................................................................................................. 24 AAV Integration .................................................................................................................. 26 Recombinant AAV Vector and Its Application for Stem Cell Transduction .................. 28
Lentiviral Vector ......................................................................................................................... 30 Adult Stem Cells ......................................................................................................................... 33 Hepatic Oval Cell ........................................................................................................................ 33
Induction and Isolation of Oval Cell .................................................................................. 33 The Origin of Oval Cells ..................................................................................................... 35 Signal Pathway of Oval Cell Activation ............................................................................ 36
Bone Marrow Cells ..................................................................................................................... 37 Hematopoietic Stem Cells (HSCs) ..................................................................................... 37
Isolation and phenotype of HSCs ................................................................................ 37 HSCs in stem cell-based gene therapy ........................................................................ 38
Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs) ....................................... 39 Isolation and in vitro characteristics of BM-MSCs.................................................... 39 Differentiation capacity and immunosupression of BM-MSCs ................................ 40
Transdifferentiation and Cell Fusion .................................................................................. 41 Adipose Tissue-Derived Mesenchymal Stem Cells (AT-MSCs) ............................................. 42
AT-MSCs vs BM-MSCs ..................................................................................................... 42 Isolation and Characterization of AT-MSCs ..................................................................... 43 Proliferation and Differentiation Capacity of AT-MSCs .................................................. 44
Liver Anatomy ............................................................................................................................ 46
2 MATERIALS AND METHODS ............................................................................................... 48
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Hepatic Oval Cell Induction and Isolation from Mouse Liver ................................................. 48 Bone Marrow Isolation ............................................................................................................... 48 AT-MSCs Isolation and Culture ................................................................................................ 49 Recombinant AAV Vector Construction and Production ........................................................ 49 Lentiviral Vector Construction and Production ........................................................................ 50 Animal.......................................................................................................................................... 51 In vitro Transduction ................................................................................................................... 51 In vivo Injection of Vectors into Mouse Liver and Muscle ...................................................... 51 Monocrotaline Treatment ........................................................................................................... 52 Adipogenic and Osteogenic Differentiation of AT-MSCs ....................................................... 52
Adipogenesis ........................................................................................................................ 52 Osteogenesis......................................................................................................................... 52
Liver Directed Transplantation of Adult Stem Cells ................................................................ 53 Immunohistochemistry for Human AAT, GFP and Mouse Albumin...................................... 53 Immunofluorescent Staining of AT-MSCs................................................................................ 54 Y-chromosome Fluorescence in situ Hybridization.................................................................. 55 Human AAT Specific ELISA ..................................................................................................... 55
3 HEPATIC OVAL CELL-BASED LIVER GENE DELIVERY .............................................. 58
Introduction ................................................................................................................................. 58 Animal Experimental Design ..................................................................................................... 58 Results .......................................................................................................................................... 59
Ex vivo Transduction Efficiency on Oval Cells by rAAV Vector .................................... 59 Lentiviral Vector Construction ........................................................................................... 59 Ex vivo Transduction and Transplantation of Oval Cells .................................................. 60
Discussion .................................................................................................................................... 61
4 EX VIVO TRANSDUCTION AND TRANSPLANTATION OF BONE MARROW CELLS FOR LIVER GENE DELIVERY OF ALPHA 1-ANTITRYPSIN ............................ 70
Summary ...................................................................................................................................... 70 Introduction ................................................................................................................................. 70 Animal Experimental Design ..................................................................................................... 72 Results .......................................................................................................................................... 73
Bone Marrow Cells Transduction ....................................................................................... 73 Liver Transplantation of ex vivo Transduced Bone Marrow Cells ................................... 73 Bone Marrow Cell Transplantation Resulted in Sustained Levels of hAAT in
Recipient Circulation ....................................................................................................... 74 Discussion .................................................................................................................................... 75
5 ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELL-BASED LIVER GENE DELIVERY ..................................................................................................................... 86
Summary ...................................................................................................................................... 86 Introduction ................................................................................................................................. 87 Experimental Design ................................................................................................................... 89
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In vivo Transduction by ssAAV and dsAAV Vectors ....................................................... 89 Ex vivo Transduction and Transplantation of AT-MSCs .................................................. 90
Results .......................................................................................................................................... 90 Isolation and Characterization of AT-MSCs ..................................................................... 90 Optimization of rAAV vecotors ......................................................................................... 91 Liver Transplantation of ex vivo Transduced AT-MSCs .................................................. 92
Discussion .................................................................................................................................... 93
6 SUMMARY AND FUTURE DIRECTION ............................................................................ 105
Summary .................................................................................................................................... 105 Future Direction ........................................................................................................................ 107
LIST OF REFERENCES ................................................................................................................. 109
BIOGRAPHICAL SKETCH ........................................................................................................... 130
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LIST OF FIGURES
Figure page 2-1 rAAV vectors construct. ........................................................................................................ 57
3-1 Experimental outline of oval cell study.. .............................................................................. 64
3-2 Flow cytometric quantification of green fluorescent oval cells after transduction of rAAV-CB-GFP vectors.......................................................................................................... 65
3-3 Construct of Lenti-CB-hAAT vectors.. ................................................................................ 66
3-4 Ex vivo transduction of oval cells.......................................................................................... 67
3-5 Detection of expression of hAAT in recipient 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 transplantation 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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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 Detection of expression of human alpha 1-antitrypsin (hAAT) in the serum.. ................ 104
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
ADULT STEM CELL-BASED GENE THERAPY FOR ALPHA 1-ANTITRYPSIN
DEFICIENCY
By
Hong Li
August 2009 Chair: Sihong Song Major: Pharmaceutical Sciences
Alpha 1-antitrypsin (AAT) deficiency is a genetic defect caused mostly by a single base
substitution in the AAT gene, and leads to hepatocyte dysfunction or lung destruction. Protein
replacement therapy is the only available treatment for AAT deficiency associated lung disease
and requires weekly repeated intravenous infusion of human AAT (hAAT) protein. For AAT
deficiency associated liver disease, no effective therapy is available except liver organ
transplantation which is limited by the shortage of donor organ. Recent studies showed that adult
stem cell gene therapy which replaces the patients’ disease-causing gene with the healthy
counterparts in their own stem cells holds great potential for the treatment of genetic diseases.
To test the feasibility of adult stem cell-mediated liver gene therapy for treatment of AAT
deficiency, we performed a series of experiments using three types of adult stem cells including
liver progenitor cells (oval cells), bone marrow (BM) cells and adipose tissue-derived
mesenchymal stem cells (AT-MSCs) for ex vivo transduction and transplantation. Using oval
cells, we confirmed the feasibility of recombinant adeno-associated virus (rAAV) vector
mediated ex vivo transduction and transplantation. Considering isolation of oval cell for
autologous transplantation is not clinically applicable, we have test the use of BM cells. We
showed that both lentiviral vector and rAAV vectors can transduce BM cells. Transplantation of
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transduced BM cells showed that BM cells transdifferentiated into hepatocytes and mediated
transgene (hAAT) expression in the liver. Importantly, sustained serum levels of hAAT were
detected in the recipient mice. Similarly, we have employed AT-MSCs since they can be
obtained easily (or less invasively) in large quantities. Results from this study demonstrated that
AT-MSCs were transduced efficiently by rAAV serotype 1 vector. After transplantation, these
cells engrafted into recipient liver, transdifferentiated into hepatocytes, contributed to liver
regeneration, and served as platform for transgene expression. Sustained serum levels of hAAT
in the recipients implied a potential application for future treatment for AAT deficiency.
AT-MSC-based gene therapy presents a novel approach for the treatment of human genetic
diseases, such as AAT deficiency. Future studies will focus on achieving therapeutic levels of
transgene expression, and gene correction in AT-MSCs for AAT deficiency associated liver
disease.
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CHAPTER 1 LITERATURE REVIEW
Introduction
Alpha 1-antitrypsin (AAT) deficiency is an autosomal recessive condition caused mostly
by a single gene mutation in the AAT coding sequence. This mutation results in abnormal
aggregation of AAT protein in hepatocytes and consequent absence of AAT protein in the
systemic circulation, which leads to hepatocyte dysfunction or lung destruction. Adult stem cell
gene therapy provides a promising treatment for long-term gene correction by taking advantage
of self-renewal and multiple differentiation potential of adult stem cells. Adult autologous stem
cell therapy also avoids the ethical quandaries presented by embryonic stem cells and the
complications from a triggered immune response.
To outline the potential of a therapy for AAT deficiency by using adult stem cells, this
chapter reviews the biology, pathogenesis, and current treatment for AAT deficiency. Current
research on recombinant adeno-associated virus (rAAV) vector, lentiviral vector and adult stem
cells (including hepatic oval cells, bone marrow cells and adipose tissue-derived mesenchymal
stem cells) are also reviewed to provide theoretical support for the adult stem cell-based gene
therapy.
Alpha 1-Antitrypsin Deficiency
AAT deficiency is a genetic disorder resulting from mutations of AAT gene. The mutation
results in a reduction of serum levels of alpha 1-antitrypsin and consequently an increased risk of
developing early onset pulmonary emphysema and severe forms of liver disease, including
cirrhosis, neonatal hepatitis and hepatocellular carcinoma.1, 2 An estimated 80,000 to 100,000
Americans are living with severe AAT deficiency, but fewer than 10 percent have been
diagnosed.3
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AAT Biology
AAT, a serine protease inhibitor, is mainly synthesized by hepatocytes and secreted into
the circulatory system with a serum concentration ranging from 20 to 53 µM. It is the second
abundant plasma protein next to albumin and has a serum half-life of 4 to 5 days.2 The AAT
molecule is a 52kD glycoprotein with 394-residue and three asparagines-linked carbohydrate
side chains.4-6 The active inhibitory site of the AAT molecule is positioned on a protrusion from
the surface of its globular molecule.7 The primary function of AAT is to protect delicate tissue
such as lung alveoli, of which major component is elastin, against the excessive proteolytic
damage of neutrophil elastase (NE) by combining the AAT inhibitory site (Met358- Ser359) with
the active proteolytic site (catalytic triad Ser173-His41-Asp88) of the protease in a mousetrap
mechanism. NE is an enzyme produced by neutrophils in response to inflammation and functions
to digest damaged tissue and bacteria. Once AAT binds NE, the AAT-NE complex remains
intact and rarely comes apart.8 As a result, AAT inhibit the activity of NE.
The AAT gene locus, designated by their protease inhibitor (PI), is located on
chromosomal segment 14q32.1 and composed of four coding exons II-V and three untranslated
exons in the 5’ region IA, IB, and IC.9 The reactive serine protease inhibitory site of AAT, Met
358 is located in exon V.7 The length of hAAT mRNA depends on the site of synthesis. In
hepatocytes, the major site of synthesis, AAT mRNA is 1.6 kb in length. AAT is also transcribed
in the minor extrahepatocyte sites such as mononuclear phagocytes and neutrophils with mRNA
of 1.8 and 2.0 kb in length.9, 10 Over 100 AAT variants have been discovered and named
according to their migration rate in a pH 4-5 isoelectric focusing gel, such as fast (F), medium
(M), slow (S), and very slow (Z). For clinical classification purposes, AAT variants may be
divided into at least four different categories: normal variants, with normal AAT serum levels
and without an association with lung or liver disease, (i.e. M variant); deficient variants, with
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reduced but detectable AAT serum concentration and associated with lung and/or liver disease,
(i.e. Z and S variants); dysfunctional variants, associated with altered function, (i.e. AAT
Pittsburgh, a thrombin inhibitor instead of a neutrophil elastase inhibitor); and null variants, with
no serum AAT and an increased risk of lung disease.11, 12
AAT Deficiency Pathogenesis
AAT deficiency was discovered in 1963 by C.-B. Laurell and S.Eriksson in Malmö,
Sweden13 when they described that three of five individuals lacking an alpha 1-globulin band in
electrophoretic analysis of serum had significant pulmonary disease. It was known that about
90% of the alpha 1-globulin band was a single protein capable of inhibiting the proteolytic action
of trypsin; hence, the term alpha 1-antitrypsin deficiency was used to define this disease state.13
After decades of AAT deficiency research, “alpha 1-antitrypsin” was recognized as a misnomer
since NE is the major target of AAT. Therefore, although it is correct that “alpha 1-antitrypsin”
inhibits trypsin, it also inhibits a number of serine proteases (proteolytic enzymes with serine at
the active site). Despite campaigns to change the name to alpha 1-antiprotease, alpha 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 are the two most common mutated alleles. The Z
allele contains a G-to-A mutation within exon V which results in a change from a negatively
charged glutamate (GAG) at position 342 to a positively charged lysine (AAG) within the
protein. This amino acid substitution causes abnormal folding and the accumulation of AAT
within the endoplasmic reticulum. The Z mutation accounts for >95% of all AAT deficiency
alleles.14 Individuals with two homozygous Z alleles have an 85% deficit in plasma AAT
concentrations and a lower association rate constant than that of the normal M allele.13 The S
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variant is an A-to-T mutation within exon III, which results in a change from glutamate (GAG)
at position 264 to valine (GTG). This resulting protein has an increased susceptibility to
intracellular degradation.15 Individuals with two S alleles have a 40% decrease in plasma AAT
concentrations.13 Consequently, AAT plasma concentrations in both ZZ homozygotes and SZ
heterozygotes are insufficient to ensure the lifetime protection of tissues from the proteolytic
damage of NE and cause clinical deficiency of AAT. Compound heterozygotes for Z allele and S
allele are also at risk if the AAT serum levels fall below the protective threshold of 11µM.16, 17
AAT deficiency is more prevalent in Caucasians, although it has been identified in virtually
every population, culture and ethnic group.18, 19 The allelic frequency of Z variant is of 0.01-0.02
in North American Caucasians and 0.02-0.03 in Northern Europeans.20
The two major clinical manifestations of AAT deficiency are lung disease, emphysema,
and liver disease (e.g. neonatal jaundice and cirrhosis). Emphysema develops when the elastin
fibers in the lung parenchyma which normally support the structure of the alveoli are destroyed
by NE. It is now known that the destruction of elastin in the lung is the result of a disturbance of
the physiologic balance between anti-protease and protease within the lung, which explains why
AAT deficiency will increase the probability of developing emphysema. There are two major
features that differentiate AAT-deficiency associated emphysema from its non-AAT deficient
counterpart. First, the age of onset is much earlier in AAT-deficient patients, for whom symptom
will present by age 35 to 50 years.21, 22 In contrast, non-AAT deficient individuals typically don’t
develop the symptoms until their 60s or 70s. Second, the disease location in the lung is more
prevalent in the lower part of the lung for AAT deficient individuals, whereas in the non-AAT
related emphysema, the disease affects the upper lung region. Cigarette smoking significantly
increases the risk and fastens the onset of AAT-deficiency associated emphysema by 10-
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15years.23 Oxidants, particularly oxidants in cigarette smoke can easily oxidize the Met358, the
active inhibitory site of AAT, and thus render AAT impotent as an inhibitor of NE.24 AAT
deficiency accounts for about 2% of the cases of emphysema seen by clinicians in the US.20
The pathogenesis of AAT-deficiency-associated liver disease is not well understood as that
of the lung disease. The mechanism by which PI*Z mutant AAT protein accumulates within the
endoplasmic reticulum of hepatocytes is likely due to the polymerization of AAT protein, a
result of a change in the three-dimensional structure secondary to the Glu342→Lys substituation.7
Aggregated mutant AAT protein is targeted for proteasome-mediated degradation. Inefficient
degradation of mutant AAT protein may predispose patients to liver disease.23 The clinical
presentation of AAT-deficiency-related liver disease is quite different from that of AAT-
deficiency-related lung disease. Only approximately 10-15% of AAT-deficient individuals with
two homozygous Z alleles will develop the liver disease23, while lung disease will occur in any
AAT-deficient individual with a serum AAT level of < 11µM. The liver disease presents in
infancy or early childhood causing progressive cirrhosis and often leading to liver failure,
whereas the lung disease presents in the early adulthood. Furthermore, the lung and liver disease
rarely coexist in the same individual.25 There is no available therapy for AAT deficiency-related
liver disease other than liver transplantation. Nevertheless, liver transplantation has a number of
important drawbacks, including shortage of donors, lifelong immunosuppressive therapy and a
significant mortality rate.
Current Therapy for AAT Deficiency
The prevention of lung disease in AAT deficiency is a relatively straightforward concept,
since the serum levels at 11µM or 800µg/ml and above is a clear indicator of having restored
anti-neutrophil elastase defense.23 Protein replacement therapy is the only FDA approved
treatment for individuals with emphysema due to AAT deficiency. The regimen of once-weekly
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infusion of human plasma-derived AAT at a dose of 60 mg/kg effectively sustains AAT serum
level above the protective threshold level of 11 µM and directly augments anti-NE protection in
the lung of individual with AAT deficiency.1 AAT protein replacement therapy appears to be
safe and well tolerated. However, there are several limitations for this therapy. One is the
potential for transmitting infectious agents in the donor’s plasma, from which AAT protein is
made. Fortunately, there has been no reported cases of blood born diseases (HIV or hepatitis)
linked to receiving this therapy. Most common reported side effects include headaches, fever,
urticaria, and fatigue, while serious side effects such as anaphylaxis and precipitation of heart
failure are rare.26 Another is the limited production capacity for AAT protein. Thousands of
doses of human AAT protein replacement have been administered, but still many patients are out
of this treatment. Alternative sources have been sought to address this shortage.
Alternative sources have been sought to address the shortage of AAT protein. A yeast-
derived recombinant AAT (rAAT) and a transgenic sheep/goat-derived AAT product have been
generated and tested in clinical trials for safety and efficacy.27-31 Unlike human AAT protein,
these rAAT products derived from yeast and transgenic animals have different glycosylation. For
example, yeast-derived rAAT lacks carbohydrate side chains 29, 32 and thus has a high renal
clearance and a much shorter plasma half-life than natural human AAT protein, which makes the
intravenous administration impractical. As an alternative, inhalation administration of
aerosolized rAAT to the lower respiratory tract of AAT-deficiency individuals has been
evaluated.33 Studies showed that aerosolize rAAT can be deposited on the alveolar epithelium
and can move from the epithelial surface into the lung interstitium, the critical site requiring
protection from NE degradation.33 Once or twice daily aerosol administration of 200mg rAAT
should result in sustained levels of anti-NE protection to the lower respiratory tract.33 Inhaled
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route of administration also reduce the dose of AAT protein in replacement therapy. The
protective threshold level in the lung is 1.2µM, 10% of the AAT plasma concentration34, and
only 2% of the intravenously infused AAT protein reaches the lung.35 For a 70kg individual, the
amount of AAT protein administrated by weekly intravenous infusion of a dose of 60mg/kg will
reduce from 4,200mg/week to 1,400mg/week for inhaled therapy, a reduction by two-thirds in
the amount.33 Unfortunately, this approach needs more clinical proof and long-term efficacy data
before it can be employed in the general population.
Gene therapy for lung disease in AAT deficiency is a direct extension of commonly used
protein replacement strategies. Besides avoiding the major concerns associated with protein
replacement (i.e. pathogen transmission), gene therapy has the capacity to produce a stable
plasma level of AAT over a prolonged period of time from a single administration. Gene therapy
also prevents the wide-range of fluctuations in AAT levels that is seen in protein replacement
therapy. Most importantly, gene therapy can provide a potential treatment for AAT deficiency
associated liver disease by implementing the strategy of delivering shRNA to correct or block the
mutant AAT gene in hepatocytes. Although the costliness of gene therapy often comes into
question, protein replacement therapy is also very expensive. The mean annual cost of protein
therapy was from $ 30,000 to $40,000, based on a dosage of 60mg/kg BW and the 1999 average
wholesale price for Prolastin®, the first marketed human AAT product, of $0.21/mg.36
Several gene therapy approaches for AAT gene delivery have been evaluated in vivo in
animal model including the non-viral delivery systems (e.g. cationic liposome, naked DNA
injection, and gene-particle bombardment delivery), and the viral delivery systems (e.g.
adenoviral, retroviral and Adeno-associated viral vector). A variety of ectopic sites (e.g.
bronchial epithelium, peritoneal surface, liver, skin and skeletal muscle) are capable of secreting
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biologically active AAT into serum.23 A phase I clinical trial evolving the intramuscular injection
of recombinant adeno-associated virus serotype 2 alpha 1-antitrypsin (rAAV2-AAT) vector in 12
AAT-deficiency adults has been done. Four dose cohorts ranging from 2.1×1012 vector genome
(VG) to 6.9×1013 VG have been tested. No vector-related severe side effects have been observed,
and especially no evidence showed that the germ line cells were infected by rAAV vectors. The
data support the general safety of this approach up to the highest dose of 6.9×1013 VG per
patient. But no therapeutic effect was detected either. A serum level of the transgene product was
detectable transiently in only one subject and the level was approximately 125-fold below the
lower end of the therapeutic range.23 Another phase I clinical trial using a rAAV serotypes 1
(rAAV1) vector for muscle delivery of AAT gene is ongoing since rAAV1 has demonstrated an
efficiency advantage of hundred-fold over rAAV2 in mouse muscle.37
Recombinant AAV Vector
AAV Genome
Adeno-associated virus (AAV) is a non-pathogenic DNA parvovirus with a linear 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 repeats (ITRs) that flank either end of AAV
genome.39 The rep gene encodes four non-structural regulatory proteins (Rep78, Rep68, Rep52,
and Rep40) by utilizing two promoters (p5 and p19) and alternative splicing. The Rep78 and
Rep68 proteins are site-specific DNA binding proteins, ATP-dependent site-specific
endonucleases, helicases, and ATPases.40 During AAV DNA replication, Rep78 and Rep68 bind
to 22-bp Rep-binding element (RBE), tandem repeats of the tetramer GAGC, and produce a 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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integration locus on human chromosome 19 to regulate viral transcription and proviral
integration.42 The Rep52 and Rep40 proteins play roles in virus assembly by generating and
accumulating single-stranded viral genomes from double-stranded replicative intermediates and
driving ssAAV genome translocation into the preformed capsid.43, 44 The cap gene encodes three
structural viral capsid proteins (VP1, VP2 and VP3) from a single promoter (p40) through a
combination of alternative splicing and alternative start codons.45 These three capsid proteins
assemble into a near-spherical protein shell of a total 60 copies of VP1,VP2, and VP3 at a molar
ratio of 1:1:10.46 The ITR is the only cis-acting elements required for viral replication, packaging
and integration.47-49 The first 125bp of ITR constitute a palindrome and fold on itself to form T-
shaped hairpin structure and the other 20 bases, called D sequence, remained unpaired.50
AAV Entry
AAV entry in target cell is not well understood yet. Current mechanism for AAV vector to
achieve transgene expression comprises receptor binding, internalization/endocytosis, trafficking
to the nuclear, uncoating, and conversion of single-stranded (ss) AAV genome to double-
stranded (ds) molecule. AAV2 gains entry into target cell by binding to primary attachment
receptor heparin sulphate proteoglycans (HSPG).51 Fibroblast growth factor receptor-1
(FGFR1)52, αVβ5 integrin heterodimers,53 and hepatocyte growth factor receptor (HGFR)54 serve
as co-receptors to facilitate the internalization. AAV2 internalizes rapidly by clathrin-mediated
endocytosis from clathrin-coated pits (half-time <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 capsid subunits necessary for priming the virus for endosomal
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 parvoviruses and has been
shown to play an important role during AAV trafficking, possibly helping AAV escape the late
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endosome.46 Point mutation in PLA2 domain results in delay onset and low level of transgene
expression.57 The ubiquitin-proteasome pathway impairs AAV intracellular trafficking.58 Recent
research demonstrated that mutating the surface-exposed tyrosine residues on AAV2 capsid
would 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 converts to the stable biologically
active ds-genome by annealing to another complementary 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.61-63 Research also has shown
the rapid uncoating of vector genome is the key to efficient liver transduction with AAV8
pseudotype vector, AAV2-based vector genome 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 second-
strand synthesis are the rate-limiting step. This explains why AAV vector demonstrates slow rise
22
in gene expression, e.g. AAV2 transgene expression is characterized by a lag phase of up to 6
weeks.64
AAV Serotype
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
existing 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 tissues66-72 since AAV serotype 2, the first AAV
serotype, was discovered as a contaminant in an adenovirus type 12 stock in the late 1960’s.67
AAV serotypes display distinct cell and tissue affinities attributed to the physical compositions
of their capsid protein coat. For example, AAV2 transduces a wide range of tissue 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 serotypes 6 and 7 are also 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 step 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, respectively, for cell surface
binding.73 The platelet-derived growth factor receptor also facilitates AAV5 entry as a co-
receptor.74 In addition, a 37kDa/67kDa laminin receptor has been identified as a receptor for
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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 capsid 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 cell 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 therapy for
alpha 1-antitrypsin deficiency37 because rAAV1 vectors have shown hundred-fold more potency
for murine muscle transduction 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 thus
transcapsidation won’t improve the transduction efficiency. Adsorption of receptor ligands to
capsid surface is to use an specific molecule e.g. bispecific F(ab’γ)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 refractory 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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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 shuffling 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 this technology. AAV-DJ show limited distribution
to the liver (and a few other tissues), superb liver performance, and the ability to evade
preexisting human immunity.84
Self-Complementary AAV
To improve AAV vector as a gene delivery tool, virus genome modification is as important
as virus capsid engineering in order to achieve desired transduction efficiency. Transgene
expression mediated by rAAV vector is not observed until 1-2 weeks post infection and reaches
to the plateau at week 4-6. The delayed transgene expression is thought to be hindered by the
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conversion of ssAAV genome to dsAAV genome either through host-cell DNA polymerase-
mediated second-strand synthesis or intermolecular hybridization between plus and minus DNA
strand, the rate-limiting step. Even though recent studies show that AAV uncoating efficacy
determines the ability of conversion from ssDNA to dsDNA.64, 85 Regardless of the mechanism,
the step can be bypassed in scAAV vector which is generated by mutating one of the AAV ITRs
to force the generation of dimeric over monomeric replicative forms, e.g. deleting the TRS
sequence from one ITR to inhibit Rep protein-mediated site-specific nicking. After uncoating,
scAAV genome folds back into dsDNA through intramolecular base pairing due to self-
complementary nature with a covalently closed ITR at one end and two open-ended ITRs at the
other. The folded molecules are ready for serving as template for transcription and result in faster
onset of transgene expression and higher transduction efficiency.86, 87 In vitro studies of 293,
Hela, and COS-7 cells show dsAAV2-CMV-GFP vector yielded 10-20% green fluorescent cells
one day after infection at a dose of 500 v.g./cell compared to less than 1% green fluorescent cells
of ssAAV2-CMV-GFP under the same condition.87 In vivo studies show 25-50% hepatocytes
displayed transgene expression 6 week after tail vein injection of dsAAV2 vector at 5×105 MOI,
but less than 5 % hepatocytes were permissive to conventional ssAAV2 vector for long-term
transgene expression.86 The important trade-off for this efficiency is the loss of half the
packaging capacity of the AAV vector with a total packaging capacity of 4.7kb, while small
coding sequence and RNA-based therapy (shRNA and microRNA, ribozymes) can be
accommodated, e.g. efficient delivery of siRNA into multidrug-resistant human breast cancer
cells to suppress MDR1 gene expression resulting in a substantial reversion of the drug-resistant
phenotype.88 What’s more, recent observation of larger genome, up to 8.9kb, being packaged
into an AAV5 capsid greatly expands the therapeutic potential of these vectors.89
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AAV Integration
After entry into the host cell nucleus, AAV can follow either lytic or latent life-cycle
depending on the presence or absence, respectively, of the helper virus such as adenovirus (Ad)
or herpesvirus (HSV). When AAV2 infects a human cell alone, its gene expression is auto-
repressed and establishes latency by integrating virus genome into human chromosome 19q13.3-
qter designated AAVS1 with a frequency of more than 70%.90, 91 AAVS1 locus (8.2kb) is near
several muscle-specific genes, p85,TNNT1 and TNNI3.90 The AAVS1 region itself is an
upstream part of a recently described gene, MBS85, whose product has been shown to be
involved in actin organization.92 Tissue culture experiment shows that AAVS1 is a safe
integration site.46
AAV site-specific integration is directed by both cellular DNA sequence and viral
components. A 33bp minimum AAVS1 sequence containing a RBE-like and a TRS-like
sequence separated by an eight-nucleotide spacer is necessary and sufficient to Rep-protein
dependent site-specific integration.93, 94 Many RBEs have been identified in human genome but
AAVS1 is the only site that has RBE and TRS in close proximity to each other.46 The AAV
components required for integration include the ITRs (in cis), Rep78/68 (in trans), and a 138bp
cis element termed the integration efficiency element (IEF), located within the P5 promoter.95 It
is proposed that Rep protein bind to REBs situated in both AAV genome and AAVS1 site to
form a Rep protein/AAV-DNA complex. Within this complex, AAV genome and AAVS1 site
are tethered to each other. Site-specific endonuclease activities of Rep78/68 introduce a strand-
specific 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
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 extrachromosomal, not integrated, genomes which are primarily responsible
for stable rAAV-mediated gene expression.97 AAV2 vectors integrate to host genome at a low
frequency of 1% in liver and no integration was undetectable in muscle (<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 end-joining (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 most 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 genes will already be expressed. However, insertional
mutagenesis by rAAV vector was observed recently in the development of hepatocellular
carcinoma in mice.100 This finding raises safety concerns on the clinical application 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
encoding 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 Coinfecting
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, Smith et al (2008) has
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shown robust and persistent transgene expression in human embryonic stem cells can be
achieved with AAVS1-targeted integration.102 A Rep-expression plasmid and an ITR flanked
EGFP reporter gene were co-transfected into hESCs using either electroporation or
Lipofectamine 2000. 4.16% of hESC clones achieved AAVS1-targeted integration. The AAVS1-
targeted hESCs retained their phenotypes and differentiated into all three primary germ layers.
EGFP expression from AAVS1-targeted clones showed significantly reduced variegated
expression and reduced tendency to undergo silencing when compared to randomly targeted
controls. In addition, transgene expression from AAVS1 locus was shown to be stable during
hESCs differentiation with more than 90% of cell expressing EGFP after 15 days of
differentiation. An insulator in AAVS1 locus was proposed to be one of the possible
explanations why the resistance to transgene silencing observed at the AAVS1 site. In addition,
the authors showed AAV ITR also impart insulation on the expression cassette in hESCs,
resulting in less variegated EGFP expression.
Recombinant AAV Vector and Its Application for Stem Cell Transduction
The first infectious clone of AAV2 was constructed in 1982 by deleting viral rep and cap
genes and inserting a transgene expression cassette between the two ITRs with Rep and Cap
genes provided in trans.103 rAAV2 has been tested in preclinical studies for a variety of diseases
such as cystic fibrosis, hemophilia, alpha-1 antitrypsin deficiency, Parkinson’s disease, muscular
dystrophy, and arthritis.104 Data on safe, broad tropism and long-term expression make rAAV
vector rapidly gain popularity in gene therapy application. Since 1995 AAV2-based vector was
first administrated to a human subject for treating cystic fibrosis,105 over 40 clinical trials have
been approved involving 14 diseases and 4 serotype rAAV vectors so far.106 These studies
indicate that in vivo gene transfer is feasible and relatively safe, but also suggest that the
transduction efficiency of AAV2 vectors fall short of requirement for adequate and organ-
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specific transgene expression. As a result, ongoing research efforts are focused on developing
new rAAV vector by modifying both AAV genome and capsid protein as described above.
Another direction of future therapy is the combination of rAAV-mediated gene delivery
with stem cell therapy. The capability of self-renewal and differentiation of stem cells makes
them to be the promising target of virus vector for long-term gene correction. Although AAV
vectors can transduce a broad range of tissues and cells, the transduction efficiency of rAAV
vector in stem cell remain further improvement. Only few researches have been shown the
successful transduction of HSCs by rAAV vectors. Santat et al. (2005) showed efficient rAAV2
transduction of primitive human cord blood HSCs (CD34+ CD38-) capable of serial engraftment
in NOD/SCID mice.107 rAAV2-transduced HSCs differentiated into all expected cell lineages
including myeloid cells and B lymphocytes after transplantation. Furthermore, transduced CD34+
stem/progenitor cells were continuously detected throughout the analysis in primary and
secondary recipients. All of these indicate that rAAV2-transduced HSCs maintain their
multipotential differentiation, long-term persistence and self-renewal capacity. Paz et al. (2007)
further demonstrated that rAAV2 vector preferentially targets quiescent subpopulation of human
CD34+ HSCs, the CD34+CD38- cell population.108 In addition, CD34+CD38- and CD34+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 human HSCs is sub-optimal levels of
expression of the cell surface receptor and co-receptor for AAV2. By applying an approach of
random 7-residue peptide library insertion into the AAV2 capsid sequence developed by Muller
et al.(2003), Sellner et al. (2008) obtained a highly efficient hematopoietic progenitor-targeted
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rAAV2 vector resulting in up to 8 fold increase on transduction efficiency of primary human
CD34+ peripheral blood progenitor cells compared to standard rAAV2 vectors.109
Unlike HSCs, rAAV can transduce mesenchymal stem cells (MSCs) efficiently. Stender et
al. (2007) showed human MSCs could indeed be transiently transduced in vitro by rAAV2
vector with efficiency of up to 65%. The transgene express reached peak at 4 days post
transduction and declined rapidly toward 0% after day 8. Transient process is ideal in the case
that temporary transgene expression is beneficial without risking potential adverse effects of
long-term transgene expression, such as the healing process. Importantly, transduced MSCs
retained multipotential activity comparable to untransduced controls demonstrated by retaining
the capability of osteogenesis, adipogenesis, and chondrogenesis.110 Ex vivo gene therapy for
osteoporosis in mouse model has shown that bone marrow-derived MSCs can be modulated to
function as continuous source of progeny osteoblasts after transduction by rAAV vector
encoding bone morphogenic protein (BMP-2), known to induce osteoblast differentiation.111
Although studies have demonstrated that supplementation of BMP-2 either by a purified protein
or through direct gene transfer into muscle can result in osteoinduction, these approaches appear
limited because of the half-life of protein and lack of target cell specificity of gene transfer
approcaches.111 However, rAAV transduction on MSCs seems to be species-specific. Rat MSCs
has been found to be refractory to transduction by AAV serotype 1-6, in contrast to rabbit MSCs
tested at the same time.112
Lentiviral Vector
Lentivirus is a member of retrovirus, a RNA virus family, of which particle consists of 2
identical single-stranded RNA molecules.113 A unifying feature of these viruses is that
replication involves the process of conversion of the viral RNA genome into double-stranded
DNA, hence the designation “retro” (backward) virus.114 Lentivirus is unique among retrovirus
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because of its ability to infect nondividing and terminally differentiated cells such as HSCs and
neurons, something that other retroviruses cannot do.115 The ability to integrate into the host
genome of nondividing cells makes lentivirus particularly attractive for permanent genetic
modification in the treatment of chronic diseases and genetic defects. Lentiviral vectors are
derived from different species such as human immunodeficiency virus (HIV-1and2)116-118,
simian immunodeficiency virus (SIV)119, feline immunodeficiency virus (FIV)120-122, equine
infectious anemia virus (EIAV)123, 124, caprine arthritis encephalitis virus (CAEV)125, and bovine
immunodeficiency virus (BIV)126, 127. Among those, the HIV-1 based lentiviral vectors are
prototypical and predominantly used in current studies116.
HIV-1 genome contains three structural genes: gag, pol and env. The gag gene encodes
three protein subunits: matrix (MA), essential for virion assembly and infection of nondividing
cells; capsid (CA), which forms the hydrophobic core of the virion and is essential for virion
assembly and maturation; nucleocapsid (NC), which coats viral RNA stochiometrically; and
several additional polypeptides of small size and unknown function, such as p1, p2 and p6. The
pol gene encodes three enzymes required for viral replication: protease (PRO), reverse
transcriptase (RT) and integrase (IN). The env gene is essential for viral binding and entry into
the host cells. It encodes the precursor glycoprotein, gp160, which is cleaved into a surface
moiety, gp120 (SU), and a transmembrane moiety, gp41 (TM). SU is required for binding to
cellular receptors and TM is responsible for the fusion with cellular membrane. Different from
simple retroviruses, HIV genome encodes two additional regulatory genes, tat and rev, and four
accessory genes, vif, vpu, vpr, and nef, all of which are involved in the viral pathogenesis.114
HIV-1 genome is flanked by long terminal repeats (LTRs) on either end. The LTR consists of
repeat region (R), unique 5’ and unique 3’ sequence at U3-R-U5 manner. Two viral integrase
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attachment sites (att) are located in the 5’ (U3) and 3’ (U5) termini of LTRs, important for
integration into host chromosome. The LTRs control viral transcriptional initiation and
termination.128
HIV-1 based lentiviral vectors are generated by co-tranfecting 3 plasmids into human cells,
including transducing vector (TV), help plasmid (HP), and vesicular stomatitis virus G (VSV-G)
envelope expression plasmid. TV contains the transgene expression cassette and cis-acting
sequences (modified lentiviral LTRs) for efficient transduction. HP comprises gag and pol gene
in order to supply reverse transcriptase and integration function in trans. HIV-1 lentiviral vector
is pseudotyped with VSV-G envelope protein to improve the tropism of HIV-1 vector.128
Promising results with lentivirial vector have been achieved in animal model for HIV-1
infection129,β-thalassaemia130, sickle cell disease131, Parkinson’s disease132, muscular dystrophy
133, etc. A phase I open-label nonrandomized clinical trial for HIV infection has been completed.
Autologous CD4+ T cells from HIV-infected subjects were transduced ex vivo with HIV-1-based
lentiviral vector, named VRX496, which contains a 937-base antisense gene against the HIV
envelope. VRX496 directly interferes with wild-type HIV (wt-HIV) expression via anti-env
antisense expression in vector transduced CD4+ T cells that become infected with wt-HIV and
thus decrease productive HIV replication from CD4+ T cells. Five subjects with chronic HIV
infection who had failed to respond to at least two antiviral regimens were enrolled. A single IV
infusion of gene-modified autologous CD4+ T cells was well tolerated in all patients. Viral loads
were stable, and one subject exhibited a sustained decrease in viral load. CD4+ T cell counts
remained steady or increased in four subjects, and sustained gene transfer was observed. There is
no evidence for insertional mutagenesis after 21-36 months of observation. Immune function
improved in four subjects134, 135. Lentiviral vectors appear promising for gene transfer to human.
33
Adult Stem Cells
An adult stem cell is defined as an undifferentiated cell found in a tissue or organ, can
renew itself, and differentiate to yield the major specialized cell types of the tissue or organ. The
primary roles of adult stem cells are to maintain and repair the tissues where they are found.
Regenerative medicine devotes to rebuilding damaged organs from stem cells including cloned
cells, embryonic or fetal stem cells, or adult stem cells. Of all the different stem cell types, only
adult stem cells might provide more medical solutions because of avoiding the ethical and legal
problems of cloning and embryonic-stem-cell approaches. Recently studies have suggested that
adult stem cells are plastic, meaning that they can differentiate not only into their original source
tissue, but also into cells of unrelated tissue.
Hepatic Oval Cells
Hepatic oval cells are bipotential progenitors that can differentiate into two types of
epithelial cells within the liver, hepatocytes and bile ductular cells, when severe hepatic injury
can not be corrected by replication of mature hepatocytes.136 Oval cells can be isolated from
animal model in large quantity and form colony and proliferate in in vitro culture supplemented
with growth facts such as stem cell factor (SCF), Flt-3 ligand, and interleukin-3 (IL-3).136
Furthermore, oval cells can differentiate into insulin-producing pancreatic cells137, and neural
cells under certain condition138.
Induction and Isolation of Oval Cell
Oval cells were first described by Kinosita et al. who observed small ovoid cells in the
livers of rats exposed to the carcinogenic azo dye ‘Butter Yellow’.139 Later on, E. Farber termed
these cells ‘oval cells’ because of their characteristic morphology, ovoid nucleus, small size
(compared to hepatocytes), and large nucleus to cytoplasm ratio.140 Oval cells didn’t invoke
attention until Thorgeirsson et al. showed that oval cells can differentiate into hepatocytes in the
34
1980s.141, 142 Under normal conditions, oval cells are quiescent and reside in the Canals of Hering
(also called cholangioles, terminal ducts, or ductules).143, 144 When severe liver damage occurs
and the proliferation of hepatocytes is blocked by exposure to hepatotoxins or carcinogens, oval
cells are activated, and proliferate in the periportal region of liver. As the liver damage
progresses, they infiltrate into the parenchyma along the bile canaliculi between the hepatic
cord.139
In rat models, the common protocol of oval cell activation employs a two-step induction
with 2-acetylaminofluorene (2-AAF) and either LD50 dose of carbon tetrachloride (CCl4) or a
two-thirds partial hepatectomy (PHx).145 2-AAF, a carcinogen, hinders hepatocytes proliferation
and PHx or CCl4 cause physical/chemical liver damage to create a regenerative stimulus.
However, mouse oval cell compartment doesn’t response to this two-step induction protocol
used in the rat model. Instead, mouse was placed on a diet containing the chemical 3, 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 oval cell accumulation after 4 to 6
weeks.147 In contrast to the rat oval cell regimen, DDC does not completely block hepatocytes
proliferation, but rather induces a chronic regenerative state in the liver.148 The DDC-induced
murine oval cells were indeed capable of liver repopulation and could rescue a metabolic liver
disease.148 Morphologically, mouse oval cells share many similarities to rat oval cells, small in
size (approximately 10µm) and a large nucleus to cytoplasm ratio; they radiate from the
periportal region forming primitive ductular structures with a poorly defined lumen. 149
To isolate oval cells from the liver, gradient density centrifugation is applied to separate
nonparenchymal compartment (NPC) fraction containing oval cells from hepatocytes after two-
step collagenase perfusion of the liver.150 To further enrich oval cells from NPC, oval cell
35
surface markers are utilized. Oval cells share molecular marker with adult hepatocyte (albumin,
cytokeratin 8 and 18), fetal hepatocytes (AFP), bile duct cell (cytokeratin 7 and 19, γ-glutamyl-
transpeptidase (GGT), OV-6 for rat and A6 for mouse).145, 151-155 Oval cell also express
hematopoietic stem cell marker (Thy-1, CD34, Flt-3 and c-kit).152, 156, 157 In rat, by using Thy-1
antibody and flow cytometric method, 95% to 97% of pure Thy-1+ oval cells can be isolated,
which also expressed the traditional oval cell markers of AFP, CK19, GGT,OC.2 and OV6152,
but were negative for desmin, a marker for Ito cells . In mice, by using Sca-1 antibody in
conjugation with magnetic activated cell sorting (MACS), more than 90% of oval cells (A6 +and
AFP +) can be enriched.149 So far, none of these protocols to induce oval cells from murine liver
would fulfill the requirements for clinical application to isolate human oval cells since they
involve administration of carcinogens. Liver progenitor cell activation has been observed in the
chronic liver diseases, such as hepatic cirrhosis due to hepatitis B158, and is also seen in the
hepatocellular carcinoma and cholangiocarcinoma development.159
The Origin of Oval Cells
The origin of oval cells has been discussed for decades but still remains controversial.
Traditionally, oval cells have been believed to originate in the liver within the Canals of Hering,
the junction between the hepatocytes canalicular system and the terminal bile ducts.139 But this
can not rule out the possibility that oval cell might be derived from other cells of either
intrahepatic origin or extrahepatic origin. Actually, most studies have implied the existence of an
undifferentiated oval cell precursor that proliferates and gives rise to oval cells.144, 160, 161
Extrahepatic origin of oval cell precursor is supported by the observation that classic
hematopoietic markers, including Thy-1, c-kit, and CD34, are also expressed by oval cells.
Additionally, sex-mismatched BM transplantation in lethally irradiated DPPIV¯ rat treated with
2-AAF/CCl4 has firstly demonstrated cells in the bone marrow were capable of repopulating the
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 cells originated from endogenous liver progenitors but not arise
through transdifferentiation from BM cells, because the oval cells isolated from the BM
transplanted mice/rats lacked the genetic markers of the original bone marrow donor.148, 163
Furthermore, the cell fusion of BM progenitors with resident hepatocytes might be the
explanation to the observation of BM-originated hepatocytes. This discrepancy might be
explained by the timing effect of exposure to hepatotoxic chemicals including monocrotalin
(MCT) and retrorsine, which plays an antimitotic activity on hepatocytes and bone marrow
cells.164 Of course, more data are needed to clarify this controversy.
Signal Pathway of Oval Cell Activation
The interaction between stromal derived factor-1 alpha (SDF-1α) and its receptor CXCR4,
a mediator of hematopoiesis165, has been proposed to be the possible mechanism by which oval
cells are activated and participate in liver regeneration.166 When oval cells are involved in the
regenerative process, it was found that oval cells expressed the stromal derived factor-1 alpha
(SDF-1α) receptor, CXCR4, while SDF-1α protein expression was up-regulated by hepatocytes
in the injured liver. Oval cells migrate along a SDF-1α chemotactic gradient to the injured liver
parenchyma. However, under non-oval cells-aided regeneration, SDF 1α expression was not
detected. SDF-1α/CXCR4 interaction possibly recruits a second wave of bone marrow cells to
the injuried liver as a percentage of oval cells are of hematopoietic origin.166 Other molecular
pathways involve the mitogenic cytokines for oval cell, i.e., tumor necrosis factor (TNF) and
interleukin-6 (IL-6). The cytokine TWEAK (TNF-like weak inducer of apoptosis) selectively
stimulates oval cell proliferation in mouse liver through its receptor Fn14 with no detectable
37
mitogenic effect on hepatocytes.167 Three primary growth factors, Hepatocyte growth factor
(HGF), epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) , are highly
upregulated in liver regeneration via the stem cell compartment. HGF acts as a strong promoter
of differentiation toward the hepatic lineage.168 Both oval cells and HSCs express the HGF
receptor c-Met.169 Transforming growth factor-beta1 (TGF-β1) and tumor necrosis factor-alpha
(TNF-α) are also being upregulated to suppress the differentiation of HSCs into megkaryocytes
and down the myeloid lineage.170, 171 These cellular factors, plus yet undetermined factors,
control the process of homing, engrafting and differentitiating into a hepatic lineage.
Bone Marrow Cells
BM is the reservoir of stem cells including two major populations, hematopoietic stem cell
(HSC) and mesenchymal stem cell (MSC). BM stem cells are one of the first stem cells to be
used successfully in humans for treating blood disease (e.g. leukemia) and BM transplantation
gave rise to the 1990 Nobel Prize in Medicine.
Hematopoietic Stem Cells (HSCs)
Isolation and phenotype of HSCs
HSCs are the best-studied and well-characterized population of stem cells mainly found in
the bone marrow. HSCs can reconstitute the whole hematopoietic system because of their
capability of giving rise to all the blood cell types including myeloid and lymphoid lineages and
their ability to replenish themselves by self-renewal. The hematopoietic tissue contains cells with
long-term and short-term regeneration capacities and mulitpotent or lineage-committed
progenitors. In human bone marrow, 1% of cells are CD34+ progenitor cells which are lineage
committed progenitor cells, including lymphoid, myeloid and erythroid progenitor cells. 0.1-1%
of total CD34+ cells are thought to represent pluripotent progenitors capable of self-renewal and
differentiation along any of the hematopoietic lineage.172 Strategies for preparing highly enriched
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HSCs may consist of combination of lineage depletion followed by positive selection of Lin-
subset that expresses specific hematopoietic markers. In lineage depletion step, cells that express
lineage markers (e.g. CD3 for T cells, B220 for B cells and some NK cells, Ly6g/Gr-1 for
granulocytes, CD11b/Mac-1 for monocyte /marcophages and TER119 for erythroid cells173) are
removed. Subsequent Lin- population is subject to positive selection for c-Kit+, Thylo, and Sca l+,
so called KTLS cells, a virtually pure population of multilineage HSCs. Thirty of KTLS cells are
sufficient to save 50% of lethally irradiated mice.173
HSCs in stem cell-based gene therapy
To date, approximately 40% of the more than 450 gene therapy clinical trials conducted in
the US have been cell-based.174 Of these, about 30% have used human stem cells, specifically
HSCs, as the means for delivering transgene into patients.175 Clinical trials have used genetically
modified HSCs to correct severe genetic immunodeficiency diseases, such as X-SCID, ADA-
SCID and CGD.176-178 These clinical trails proved the concept of stem cell-based gene therapy
but also pointed out potential barriers to the development of the treatments using HSCs. First,
gene transfer into HSCs did not lead to efficient transduction rates of these cells. Second, over
time, the transgene get turned off, known as gene silencing, due to cellular mechanisms that alter
the structure of the area of chromosome where the therapeutic gene has been inserted.179-183
Third, HSCs can not be expanded ex vivo.
In recent years, scientists have overcome some of these limitations. To improve the
transduction efficiency of HSCs, virus vectors that can transduce non-dividing cells (e.g. HSCs)
have been explored, such as lentiviral vector and AAV. Another approach has been to stimulate
HSCs to divide without differentiating by using cytokines, i.e., flt3-ligand and stem cell factor.
The inability to expand HSCs ex vivo hinders the current application of HSCs in both cell and
cell-based gene therapy. This is especially true in cases where the number of available stem cells
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
cells.162, 185, 186 These observations implies even broader applications of HSCs in both cell and
stem cell-based gene therapy.
Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs)
MSCs are a heterogeneous population of plastic-adherent, spindle-shaped and 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, MSCs are able to form fibroblastic colonies, termed colony-forming unit-fibroblasts
(CFU-F). As another distinct stem cell population which resides in bone marrow, MSCs are a
population of stem cell responsible for the maintenance of non-hematopoietic bone marrow
elements which promote HSC proliferation and differentiation. The history of MSCs can be
traced back 130 years ago when the German pathologist Cohnheim first suggested the presence
of non-hematopoietic stem cells in his study of wound repair.187 It was not until the mid-1970s
that Friedenstein and his colleagues first successfully isolated fibroblast-like cells from bone
marrow.188
Isolation and in vitro characteristics of BM-MSCs
To date, Friedenstein’s procedure of MSCs preferential attachment to tissue culture plastic
is still considered as the standard approach to isolate MSCs. Marrow aspirate, from the tibias and
femurs of the rodent experimental animals or the iliac crest of human, is applied to density
gradient centrifugation to isolate the BM mononuclear cells (BM-MNCs). BM-MNCs are plated
on the tissue culture plastic in medium with 10%FBS. MSCs representing approximatly1 in
10,000 BM-MNCs attach and grow as fibroblastic cells that develop into visible symmetric
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 investigated to
enrich more homogenous population of MSCs. The approach of negative selection for MSCs
lacking the expression of endothelial marker (e.g., CD31) and HSC maker (e.g., CD34,CD45,and
CD 14) is more widely used. 191
The in vitro cultured MSCs are both morphologically heterogeneous, ranging from narrow
spindle shaped cells to large polygonal cells, and phenotypically heterogeneous due to culture
medium, plating density, and species from which MSCs are isolated. In general agreement,
MSCs do not express either hematopoietic markers CD 34, CD45, CD14, and CD11 or
endothelial markers CD31, but they do express stromal-associated marker CD105 (SH2), CD73
(SH3/4), CD44, CD90, CD71 and Stro-1 as well as the adhesion molecules CD106 (VCAM-1),
CD166 (ALCAM), ICAM-1 and CD29.190 Unfortunately, no single unique marker is specific to
MSCs. Multilineage potential is another important criterion for identifying the putative MSC
population. Though not immortal, MSCs can expand in vitro many-fold and still retain the multi-
lineage potential. By culturing MSCs under conditions that are favorable for adipogenic,
chondrogenic or osteogenic differentiation for 1 to 3 weeks, MSCs were highly differentiated
without evidence of the other lineages.189 In contrast, cultured primary fibroblasts, the mature
mesenchymal cells, don’t undergo any such differentiation under same induction condition.
Differentiation capacity and immunosupression of BM-MSCs
In addition to differentiation into its native derivatives, the mesenchymal tissue such as
bone, cartilage, adipose, tendon, and muscle, MSCs have the potential to differentiation into
other cell types such as hepatic, renal, cardiac, and neural cells 192-198. Both Toma et al. (2002)
and Barbsh et al. (2003) demonstrated that MSCs can migrate and engraft in infracted
myocardium and appeared to differentiate into cardiomyocytes after either site-directed or
41
systemic delivery of MSCs. 193, 194 The two-step protocol with the use of hepatocytes growth
factor and oncostatin M has been developed to in vitro effectively induce hepatic induction of
MSCs. Differentiated MSCs gained cuboidal morphology of hepatocytes and expressed liver
specific marker gene in a time-dependent manner. Differentiated cells further demonstrated in
vitro functional characteristic of liver cells including albumin production, glycogen storage, urea
secretion, uptake of low-density lipoprotein and cytochrome P450 activity.198 Hepatic-
preconditioned human MSCs functionally integrated into hepatectomized mouse liver
administered by intrasplenic injection.195
An important advantage of using MSCs is that in some situation they don’t need to be
matched, since the immune phenotype of MSCs (widely described as MHC I+, MHC II-,CD40-
,CD80-, and CD86-) is considered as non-immunogenic; therefore transplantation into an
allogeneic host may not induce immunoresponse.190 This provides the possibility to use MSCs as
an “off-the-shelf” product. Furthermore, MSCs exhibit immunomodulatory and anti-proliferative
effects on T cells. When MSCs are present in mixed lymphocyte culture (MLT), T-cell
proliferation is suppressed in a dose-dependent fashion.199-201 A phase II clinical trial has been
conducted by using ex vivo expanded MSCs to treat patients with steroid-resistant, severe, acute
graft-versus-host disease (GVHD) after hematopoietic-stem-cell transplantation. For 55 patients,
30 patients lost all the symptoms of acute GVHD and 9 patients showed improvement.202
Transdifferentiation and Cell Fusion
Transdifferentiation, the conversion of adult stem cells from one lineage to another, has
been proposed to explain the observation that transplanted bone marrow stem cells can turn into
unexpected lineages such as myocytes, endothelial cells, hepatocytes, neurons and many others.
145, 203-205 If this concept, transdifferentiation, is true, it would remove the need to collect the stem
cells from embryo and thus avoiding many of the political and ethical barriers to stem cell
42
therapy. In order to identify the molecules that are responsible for reprogramming the adult stem
cells to acquire these new lineages, Terada et al. (2002) and Ying et al. (2002) set out to show
that embryonic stem (ES) cells can induce the BM stem cells or neural stem cells to
transdifferentiate into embryonic-like plurioptent stem cells by culturing them with embryonic
stem cells in vitro, respectively. Instead, they found that ES cells and BM cells fused with each
other to create tetraploid cells which carried markers for both the ES cells and the adult stem
cells206, 207. It therefore started a new debate on whether cell fusion, rather than
transdifferentiation, is responsible for adult stem cell plasticity. However, cell fusion can not
explain all the cases of transdifferentiation. Adult stem cells can adopt new fates in vitro when
ES cells are not present, suggesting the enviromental influences can switch cell lineage. Zhang et
al. (2004) also demonstrated that cell fusion and transdifferentiation may account for the
transformation of peripheral blood CD34+ cells into cardiomyocytes in vivo.208 In view of the
greater aims of gene therapy, as long as the resulting cells are healthy and functional, either
transdifferentiation or cell fusion can be used as a means for stem cell gene therapy in which
adult stem cells serve as vehicle. Nonetheless, more studies are needed for the future of stem
cell-based regenerative medicine.
Adipose Tissue-Derived Mesenchymal Stem Cells (AT-MSCs)
AT-MSCs vs. BM-MSCs
MSCs have traditionally been isolated from bone marrow aspirates, although recent studies
have reported that MSCs can be isolated from other tissues such as cord blood, peripheral blood,
fetal liver, and adipose tissu0065.209-212 Among these tissues, adipose tissue presents a promising
stem cell resource of repeatable access, replenishment, easy isolation and minimal patient
discomfort. There is little to no difference between BM derived-MSCs and AT-MSCs regarding
the morphology, immune phenotype, yield of adherent stromal cells, growth kinetics, cell
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, the colony frequency is higher in adipose tissue than in bone marrow e.g. the
number of CFU-F was 557±673 for adipose tissue vs. 83±61 for bone marrow at an initial
plating density of 1×106 cells per cm2, as well as the maintenance of proliferating ability in
culture.197, 215, 216 Harvesting of BM is also a highly invasive procedure and the number,
differentiation potential, and maximum life span of MSCs from BM decline with increasing age
217-219. Although the attachment and proliferation capacity are more pronounced in AT-MSCs
derived from younger donors compared with older donors, the differentiation capacity is
maintained with aging.220 Taking all of these issues into account, adipose tissue might be a more
attractive alternative to BM in isolating MSCs.
Isolation and Characterization of AT-MSCs
Adipose tissue, like bone marrow, is derived from embryonic mesoderm and contains a
heterogeneous stromal cell population including a putative stem cell population.210
Approximately 400,000 liposuction surgeries are performed in the US each year and these
surgeries yield anywhere from 100ml to >3L of lipoaspirate tissue.221 Recent researchers have
found the liposuction waste are an alternative source of adult stem cells that are believed to
contribute to repair and healing as regenerative medicine for tissue engineering. At least 5
different types of adipose tissue exist: bone marrow adipose tissue, brown adipose tissue,
mammary adipose tissue, mechanical adipose tissue, and white adipose tissue. Each adipose
tissue type serves a distinct biological function.214 Of great interest to regenerative medicine is
the stem cells-derived from white adipose tissue, which can differentiate along multiple
pathways in vitro.222
44
Rodbell and Jones presented the first in vitro isolation method for mature adipocytes and
progenitor cells from the rat fat pad in the 1960s.221 Tissue was minced into small fragments,
digested with collagenase Type I at 370C, and fractionated by differential centrifugation. The
supernatant contained the mature adipocytes which floated due to their high lipid content. The
pellet contains the stromal vascular fraction (SVF) which consisted of circulation blood cells,
fibroblasts, pericytes, endothelial cells, adipose progenitors and putative stem cells. SVF was
plated in the tissue culture plastic to enrich the plastic adherent population. The mean number of
nucleated SVF cells was determined at 308,849 cells per ml of lipoaspirate tissue and the initial
nucleated SVF cells contained colony-forming unit fibroblasts at a frequency of 1:32.222 Besides
AT-MSCs, a variety of names have been used to described the fibroblast-like, plastic adherent,
multilineage potential cell population isolated from collagenase digests of adipose tissue, such as
adipose-derived stem/stromal cells (ASCs), adipose-derived adult stem (ADAS) cells, adipose
mesenchymal stem cells (ADMSCs), and processed lipoaspirate (PLA) cells, etc. The surface
immunophenotype of human AT-MSCs resembles that of human BM-derived MSCs, with >
90% -identity based on direct comparison221, that is negative to endothelial marker ( CD144) or
HSC maker ( CD34, CD45, CD 14) but positive to typical MSC marker (CD29,CD44,CD73,
CD90,and CD105).197 Discrepancy has been observed in published reports due to the lack of
consistency between laboratories with respect to the isolation and cell passage. The
immunophenotype of AT-MSCs progressively change with passage such that classic stromal cell
markers presents only on 0.8%-54% of the initial SVF, and on up to 98% of AT-MSCs at late
passage.222
Proliferation and Differentiation Capacity of AT-MSCs
Following isolation, human AT-MSCs remain inactive with an initial lag time of 5-7 days,
and then enter a proliferative phase, reaching confluence within 48hrs.210 Human AT-MSCs
45
display a cell doubling time of 2 to 4 days depending on culture medium and passage number222,
223 or 60hrs in average under standard culture condition (i.e., 10%FBS)210. In vitro culture, MSCs
demonstrate a limited life span and finally undergo replicative arrest or senescence demonstrated
by loss of proliferation and altered morphology.197 Izadpanah et al. (2006) showed that human
AT-MSCs can be expanded routinely beyond 180-210 population doublings223, greater than the
upper limit of most somatic cells (80 population doublings)224. With prolonged passage for > 4
months, human AT-MSCs have been observed to undergo malignant transformation
characterized by abnormal karyotype and tumor formation when implanted into immunodeficient
mice, which might be due to the overexpression of oncogene c-myc. 225
Not only can AT-MSCs differentiate into mesodermal lineages e.g., adipogenic,
osteogenic, chondrogenic, and myogenic cells but also into ecto- and endodermal lineages as
well (e.g., neurons, endocrine pancreatic cells, hepatocytes, endothelial cells, and
cardiomyocytes). 202, 226-231 Evidence that AT-MSCs can be applied as a source of hepatocytes is
the observation that the human CD105+ fraction of AT-MSCs reveal several liver-specific
markers and functions, such as albumin production, low density lipoprotein uptake, and
ammonia detoxification, after treatment with a hepatic induction growth factor cocktail
(HIFC).232 More importantly, CD105+ AT-MSC-derived hepatocyte-like cells can be
transplanted and incorporated into the host liver parenchyma. In addition, AT-MSCs support
complete differentiation of hematopoietic progenitor into myeloid and B lymphoid cells.233 This
suggests that the addition of AT-MSCs infusion may improve and accelerate hematopoietic
stem-cell engraftment in recipients who have undergone BM ablation. The molecular mechanism
of the lineage-specific differentiation into cells and tissues of mesodermal origin is well
documented in the review paper by Schaffler and Buchler (2007). Yet, the molecular event
46
behind the “cross-differentiation” is far from clear and more research is needed to decode this
puzzle for future AT-MSC-related cell therapy and tissue engineering. For clinical purposes,
adipose tissue derived stem cells might appear superior to bone marrow cells in view of their
availability in large quantities at a low risk to patients.
Liver Anatomy
Liver is the largest gland in the body consisting of several separate lobes and accounting
for 2% of the body weight in the human and 5% in the mouse.234 It is the only organ with two
separate afferent blood supplies. Hepatic artery provides oxygenated blood and the portal vein
supplied the liver with venous blood rich in nutrients and hormones from the intestines and
pancreas. Roughly 75% of the blood entering the liver is venous blood. Arterial and venous
blood mix as they enter the sinusoids, distensible vascular channels lined with discontinuous and
fenestrated endothelial cells and bounded by hepatocytes, in the liver. When blood flows through
the sinusoid, oxygen, carbon dioxide, nutrients, proteins and wastes are exchanged between
blood and hepatocytes, and finally blood empty into the central vein. Hepatocytes are arranged in
cords, cellular arrays of one-cell-wide, radiating from the central vein with their basal surfaces
facing and surrounding the sinusoids and the apical faces of adjoining hepatocytes form
canaliculi. Bile secreted by the hepatocytes is collected in the canaliculi and flow parallel to the
sinusoid, but in the opposite direction to the blood flows. At the end of the bile canaliculi, bile
drains into bile ducts that lie in very close proximity to the terminal branches of the portal vein
and hepatic artery. Collectively, these three structures are called the portal triad.
The main cell type of the liver is parenchymal cells including hepatocytes and bile duct
epithelia. Non-parenchymal cells of the liver include kuffer cells (marcophages in the hepatic
sinusoid), stellate cells (located under the sinusoid), vascular endothelial cells, and pit cells
(natural killer cells). Among these, hepatocytes are responsible for the majority of liver function,
47
and constitute ~60% of the liver cell population and 90% of liver mass. An adult mouse liver
contains about 5×107 hepatocytes, and an adult human liver contains about 8×1010
hepatocytes.235 Hepatocytes are the largest and polygonal-shaped epithelial cells (30-40µm) with
diploid or tetraploid nuclei and a large proportion of adult hepatocytes are binucleated.236 Zone-
1,-2 and -3 hepatocytes are distinguished based on the basis of their relative position within the
lobule. Zone-1 hepatocytes surround the portal triad, zone-3 hepatocytes are around the central
vein, and zone-2 hepatocytes are the inter-zonal hepatocytes. Hepatocytes at different zone are
heterogeneous in the size and metabolic /biosynthetic function, having smaller and usually
single-nucleated hepatocytes in zone 1 and predominantly bi-nucleated hepatocytes in zone-3.
Liver performs a variety of biochemical functions including the metabolism of amino
acids, lipids, and carbohydrates, the detoxification of xenobiotics, the synthesis of serum proteins
and secretion of bile. Additionally, liver has an astounding capacity to regenerate after injury.
Animals (including humans) can survive surgical removal of up to 75% of the liver mass.237 The
residual lobes enlarge to make up for the removed mass, although the resected lobes never grow
back. The original number of cell is restored within 1 week and the original tissue mass with 2 to
3 weeks.236, 238 Parenchymal regeneration after the surgical loss of liver tissue principally
originates from the proliferation of the remaining mature hepatocytes rather than from liver
stem/progenitor cells. Typically 10-12 hrs after partial hepatectomy (PHx), hepatocytes in the
remaining liver initiate the DNA synthesis which peaks at 24-48 hrs depending on the species237.
Hepatocyte proliferation starts in the periportal area and proceeds to the pericentral area237.
Following the hepatocytes, the other hepatic cell types enter into DNA synthesis 24 hours later.
48
CHAPTER 2 MATERIALS AND METHODS
Hepatic Oval Cell Induction and Isolation from Mouse Liver
Adult male C57BL/6 mice (8 week old) were fed with a diet containing 0.1% DDC
(BioServe, Frenchtown, NJ) for 4 -6 weeks. This diet has been approved to be very effective for
inducing and enriching murine oval cells in liver.146 A two-step collagenase perfusion was
applied for hepatocyte and nonparenchymal (NPC) isolation.150 Low –speed centrifugation (500
g) separated NPC fraction containing oval cells from hepatocytes. The oval cells were further
enriched by magnetic activated cell sorting using Sca-I antibody conjugated magnetic beads
(Miltenyi Biotec, Germany).
Bone Marrow Isolation
BM cells were isolated from the femurs and tibias of C57BL/6 male mice. The bone was
sterilized by immersion in 70% ethanol, and the skin and muscles were removed. BM was
exposed by cutting the ends of the bones, and extruded by inserting a 20-gauge needle attached
to a 3ml syringe and forcing 1-2 ml of DMEM (Mediatech, Inc., Manassas, VA) containing 2%
FBS (HyClone laboratories, Inc., Logan, Utah) through the bone shaft. To make a single cell
suspension, BM was triturated by gently aspirating several times using the same needle and
syringe and passed through a 70µm nylon mesh strainer (Becton Dickinson Labware, Franklin
Lakes, NJ). Cells were treated with red blood cell (RBC) lysing buffer for 2 minutes at room
temperature to deplete RBC. After that cells were cultured for 1h at 37℃ to remove the
macrophages which will attach to the bottom of the cell culture dish. Those remaining
suspension cells contained mesenchymal and hematopoietic stem cells, and blood progenitor
cells and were ready for virus vector transduction. Transduction was performed in a 1ml reaction
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 resuspended in 100ul saline for transplantation or cultured in
murine myeloid long-term culture medium (MyeloCultTM M5300, StemCell Technologies Inc.,
Vancouver, BC) for in vitro transduction efficiency study.
AT-MSCs Isolation and Culture
Mouse AT-MSCs were isolated from peritoneal adipose tissue excised from the abdominal
region of 6 to 8-week-old male C57BL/6 mice. Adipose tissue was enzymatically digested with
0.075% collagenase (type I; Sigma-Aldrich, St. Louis, MO) in PBS for 1hr at 37°C with gentle
agitation. The collagenase was inactivated with an equal volume of DMEM (Mediatech, Inc.,
Manassas, VA) supplemented with 10% fetal bovine serum (FBS, HyClone laboratories, Inc.,
Logan, Utah), and the infranatant was centrifuged at 1,000 rpm for 5 min at room temperature.
The cell pellet was resuspended in 160mM NH4Cl (StemCell Technologies Inc, Vancouver,BC)
and incubated at room temperature for 2 min to eliminate contaminating red blood cells and
filtered through a 100-µm nylon mesh strainer (Becton Dickinson Labware, Franklin Lakes, NJ)
to remove debris. The resulting AT-MSC-containing cell pellet is collected by centrifugation as
described above, and resuspended in a DMEM/10%FBS medium at 1-2×106 cells/100 mm
plastic tissue culture dishes. Non-adherent cell population was poured off after 12-16hr culture.
Adherent cells were washed with PBS and cultured in DMEM with 10% FBS for expansion. The
cells with 70-80% confluence were harvested with 0.25% trypsin-EDTA and reseeded at 1.0×105
cells/60mm dish.
Recombinant AAV Vector Construction and Production
Recombinant single-stranded AAV vectors used in this study has been described
previously.239, 240 Briefly, plasmid ssAAV-CB-hAAT contained full length AAV2 ITRs, hAAT
50
cDNA flanked by two ITRs and driven by CMV enhancer/chicken-β-Actin promoter, intron and
ploy(A) sequence (Figure 2-1A). dsAAV vector construction was described previously.87 In
brief, dsAAV vector plasmid was made from the ssAAV-CB-hAAT plasmid by deleting 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 was replaced by
duck hepatitis B virus (DHBV) promoter (Figure 2-1B) or CMV promoter (Figure 2-1C).
Plasmid ssAAV-CB-hAAT, dsAAV-DHBV-hAAT, and dsAAV-CMV-hAAT were packaged
into AAV serotype 1 and 8 capsids, respectively, as described previously.241 In brief, , vector
plasmid was co-transfected with helper plasmid(s) which contain genes from adenovirus and
corresponding serotype AAV cap and rep genes into 293 cells. Cells were harvested and
disrupted by freeze-thaw lysis to release virions. The rAAV vectors were purified by iodixanol
gradient centrifugation followed by heparin affinity or anion exchange chromatography. The
physical particle titers of vector preparations are routinely assessed by quantitative dot blot
analysis.
Lentiviral Vector Construction and Production
In order to generate a lentiviral vector expressing hAAT (Lenti-CB-hAAT), hAAT cDNA
fragment and CB promoter was released from plasmid AAV-CB-hAAT and inserted into pTYF-
linker vector which was derived from an LTR-modified recombinant HIV-1 plasmid. This
lentiviral vector was packaged by as previously described. Briefly, pHP-helper (packaging helper
construct), pHEF-VSV-G (envelop expression construct) and pLenti-CB-hAAT (transducing
construct) were cotransfected into 293T cells using superfect reagent (Qiagen Inc.). The
lentiviral vector was harvested at 48hr after transfection by collecting the cell culture medium
and was concentrated by centrifuging at 2500g under 4℃ for 20 minutes x 2times using the
51
Amicon Ultra-15 centrifugal filter device (Millipore). The virus titer was estimated at 1×109 viral
particles/ml.242
Animal
C57BL/6 mice were purchased from Jackson Laboratory, housed in a specific pathogen-
free room. All animal work was conducted under the protocols approved by the University of
Florida Institutional Animal Care and Use Committee.
In vitro Transduction
Cells were seeded in 24-well plates (Costar, Corning Inc.) with 1×104cells/well and
infected with virus vectors at different multiplicities of infection (MOI) in triplicate. Oval cells
were cultured in Iscove’s Modified Dulbecco’s medium (StemCell Technologies,Inc.,Vancouver,
BC) with 10% FBS, 1000 I.U./ml penicillin and streptomycin, 100ng/ml IL-6, 100ng/ml Flt-3,
100ng/ml SCF, and 20ng/ml GM-CSF. All these growth factors were purchased from StemCell
Technologies (Vancouver BC). BM cells and AT-MSCs were culture in DMEM medium
(Mediatech, Inc., Manassas, VA) with 10% FBS and 1000 I.U. /ml penicillin and streptomycin.
The accumulative hAAT secretion in the culture medium was collected and measured by hAAT
specific ELISA.
In vivo Injection of Vectors into Mouse Liver and Muscle
For muscle injection, 8-week-old female C57BL/6 mice were anesthetized by isoflurane
inhalation, and aliquots of vectors were injected percutaneously into the quadriceps femoris
muscles of both hind limbs.243 For portal vein injection, mice were anesthetized by isoflurane
inhalation and a ventral midline abdominal incision was made into the peritoneal cavity to
expose the portal vein.239 Vectors were injected into the portal vein using a 30-gauge needle.
Hemostasis was achieved by application of a small piece of cotton directly onto the injection site.
The volume of vector was 100ul and the total amount of virus injected per mouse is 2×1010
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 acidified PBS
(pH 3.0) using 2N HCl by gentle stirring. After complete dissolution, solution was adjusted to
pH7.0 with 5N NaOH and additional PBS was added to achieve total volume of 10ml and final
concentration of 50mg/ml. The standard MCT treatment consisted of two intraperitoneal
injection of MCT at 50mg/kg BW with a 2-week interval. After the final injection, mice were
housed for two more weeks before further studies were conducted.
Adipogenic and Osteogenic Differentiation of AT-MSCs
Adipogenesis
At passage 3, AT-MSCs were seeded in 6-well plate and grown to 100% confluence for
differentiation. Adipogenic differentiation was induced by culturing cells in the adipogenic
medium for 2 weeks with medium changes twice weekly. Adipogenic medium consists of
DMEM supplemented with 10% FBS, 0.5mM 3-isobutyl-1-methylxanthine, 1µM
dexamethasone, 200µM indomethacin, and 10 µg/ml bovine insulin (all Sigma). Adipogensis
was assessed by staining for intracellular lipid droplets with Oil Red O stain (Sigma).
Osteogenesis
Cultured cells at passage 3 were seeded in 6-well plate and grown to 100% confluence for
differentiation. Osteogenic differentiation was induced by culturing cells in the osteogenic
medium for 2 weeks with medium changes twice weekly. Osteogenic medium consists of
DMEM supplemented with 10% FBS, 0.1µM dexamethasone, 10mM β-glycerophosphate, and
53
50 µM ascorbate-2-phosphate (all Sigma). Osteogenesis was assessed by staining for calcium
depositions with Alizarin Red S (pH 4) stain (Sigma).
Liver Directed Transplantation of Adult Stem Cells
Adult female C57BL/6 mice received MCT (50mg/kg BW) at a two-week interval by i.p.
injection for inhibiting the endogenous liver cell proliferation.245 Two weeks after second
injection, the mice were partially hepatectomized to remove 70% liver (large median and left
lateral lobes of the liver) under general anesthesia.246 In the meantime, adult stem cells
suspended in 100ul saline were transplanted into the remaining liver immediately after PHx by
portal vein injection as previously described.239 To transplant adult stem cells by intrasplenic
injection, cells were injected into the inferior tip of the spleen of mice right after PHx using a 30-
gauge needle. To aid in the coagulation process, splenic injection site was ligated using sterile
absorbable surgical suture (Ethicon, INC., Somerville, NJ). Post-surgery, mice were placed back
in specific pathogen-free room. Blood sample was collected from the tail vein each week. At the
end of experiment (8-14 weeks post surgery), samples of liver, lung, kidney, ovary, pancreas,
brain, heart, intestine, and bone were collected and subjected to OCT embedding or Paraffin
embedding.
Immunohistochemistry for Human AAT, GFP and Mouse Albumin
Organ tissues were fixed in 10% neutral buffered formalin (NBF) and embedded in
paraffin. For hAAT and GFP immunostaining, tissue sections (5μm) were de-paraffinized,
rehydrated, and blocked for endogenous peroxidase with 3% hydrogen peroxide in methanol for
10 minutes. To detect hAAT expression, tissue sections were incubated with primary antibody,
rabbit anti-human AAT (1:800, RDI/Fitzgeral Industries, Concord, MA, USA), for overnight at
4°C. Staining was detected using ABC-Rb-HRP and DAB kits (Vector laboratories, Burlingame,
CA). Antigen retrieval was performed in Digest-All™ (trypsin) (Zymed® Laboratories,
54
Carlsbad, CA, USA) for 5 minutes at 37°C, followed by incubation in Trilogy (Cell Marque
Corp., Rocklin, CA, USA) for 25 minutes at 95°C. Immunostaining of GFP was performed
using rabbit anti-GFP antibody (1: 10,000, AbCam Ltd, Cambridge, MA). Staining was detected
using ABC-Rb-HRP and DAB kits (Vector laboratories, Burlingame, CA). Antigen retrieval was
performed in Trilogy (Cell Marque Corp., Rocklin, CA, USA) for 25 minutes at 95°C. To detect
albumin expression, antigen retrieval was performed using citrate retrieval for 30 minutes in a
steamer. The tissues were incubated with goat anti-mouse albumin (1:5,000, Abcam, Ltd.,
Cambridge, MA) overnight at 4°C, followed by incubation with biotinylated horse anti-goat
(1:200, Vector Laboratories) for 30 minutes. Staining was developed by Vectastain ABC-
Alkaline Phosphatase kit (Vector Laboratories) Vulcan Fast Red (VFR) chromagen (Biocare
Medical, Concord, CA, USA). Immunofluorescence double staining for human AAT and GFP
was performed as previously described with minor modification.245 Co-localization of hAAT and
GFP were detected by staining sequentially with anti-GFP (1:500, AbCam Ltd, Cambridge, MA)
and anti-hAAT (1:100, RDI/Fitzgeral Industries, Concord, MA, USA). The FITC (1:1000) - or
rhodamine (1:1000)-conjugated secondary antibody were applied.
Immunofluorescent Staining of AT-MSCs
The cells were plated onto glass chamber slides for a day, and then fixed for 15 min in 4%
paraformaldehyde in 100mM sodium phosphate buffer (pH 7.0). The cells were washed for 10
min in 100mM glycine in PBS (PBS/glycine) and blocked for 1 h in immunofluorescent blocking
buffer (IBB) containing 5% bovine serum albumin (BSA), 10% FBS, PBS, 0.1% Triton X-100.
The cells were subsequently incubated for 1 h in IBB containing the following anti-mouse
monoclonal antibodies: CD31, CD34, CD44, CD45, CD90, CD105, and CD133 (1:100,
eBioscience, San Diego, CA). The cells were washed extensively with PBS/glycine and
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 Fluorescence in situ Hybridization
5μm sections of paraffin embedded liver tissue samples were used for detecting Y-
chromosome. Liver sections were de-paraffinized 2×5 min in fresh xylene and rehydrated 2×2
min in 100% ethanol, 2min in 95% ethanol, 1 min in 70% ethanol and 1 min in water. 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 2×SSC
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/2×SSC, 2×SSC, and 4×SSC+0.1% Igepal (NP-40) at 460C for 7
min, respectively. For detection, air dry slide at RT in dark and mount glass coverslips with
DAPI (Vector, Burlingame, CA).
Human AAT Specific ELISA
Microtiter plates were coated with 100ul of goat anti-hAAT (1:200, Sigma
Immunochemical, St.Louis, MI, USA) in voller’s buffer overnight at 4oC. Blocking buffer, 3%
bovine serum albumin (BSA, Sigma, St.Louis,MI,USA), was added to saturate the remaining
sites for protein binding on the microtiter plate and incubate 1 hour at 37oC. After blocking,
duplicated standard curves (Sigma Immunochemical, St.Louis,MI,USA) and diluted sample
serum or cell culture medium were loaded and incubated 1hr at 37oC. A second antibody, rabbit
anti-hAAT (1:1000, Roche Molecular Biochemicals, Indianapolis,IN, USA),was added and
56
reacted with captured hAAT at 37oC for 1 hour. A third antibody, goat anti-rabbit IgG
conjugated with peroxidase (1:80, Roche Molecular Biochemicals, Indianapolis,IN, USA) was
incubated at 37oC for 1 hour. The plate was washed with PBS-Tween 20 three times between
reactions. After reacting with the substrate (o-phenylenediamine, Sigma Immunochemical,
St.Louis, MI, USA), Microtiter plate was read at 490nm on a MRX microplate reader (Dynex
Technologies, Chantilly, VA, USA).
57
A
ITR CB promoter hAAT pA ITR
B
Delt-ITR DHBV promoter hAAT pA ITR
C
Delt-ITR CMV promoter hAAT pA ITR
Figure 2-1. rAAV vectors constructs. (A) rAAV-CB-hAAT. Plasmid rAAV-CB-hAAT contained
full length AAV2 ITRs, hAAT cDNA driven by CMV enhancer/chicken-β-Actin promoter, intron and ploy(A) sequence. (B) rAAV-DHBV-hAAT. D-sequence and terminal resolution site (trs) of 5’-ITR is deleted to make self-complementary dsAAV vector. 3’-ITR remains intact. hAAT is driven by DHBV promoter. (C) rAAV-CMV-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.
58
CHAPTER 3 HEPATIC OVAL CELL-BASED LIVER GENE DELIVERY
Introduction
Our previous study has shown that hepatic oval cells can be transduced by rAAV vectors
and transplanted into the recipient liver.245 Transgene (hAAT) was expressed in the engrafted
donor cells and transgene product (hAAT protein) was secreted into the circulation of recipient.
The transgene expression was also sustained throughout the experiment, 14 weeks post
transplantation. Results from previous studies formed solid base for future investigations of adult
stem cell transplantation studies in a model for adult. However, the level of the transgene
expression remain improvement.247 The goal of this study is to further evaluate the potential of
viral vector-mediated adult stem cell-based therapy. Since transgene expression is affected by
transduction efficiency of viral vector, transplantation efficiency, and engraftment capability of
adult stem cells, we have tested the possibility of enhancing oval cell transduction efficiency by
optimizing rAAV vectors and employing a lentiviral vector.
Animal Experimental Design
Freshly isolated liver oval cells (2×106 cells) from male C57BL/6 mice were infected with
rAAV1-CB-hAAT at 1×104 vg/cell or Lenti-CB-hAAT at 5vg/cell, respectively, for 2 hr, washed
with PBS three times, and transplanted into partially hepatectomized female C57BL/6 mouse
liver (n=5) by intrasplenic injection. Donor mice were treated with a diet containing 0.1% diethyl
1, 4-dihydro-2, 4, 6-trimethyl-3, 5-pyridine-dicarboxylate (DDC) for 4 weeks to stimulate oval
cell proliferation. Recipient mice were injected with monocrotaline (MCT) 50mg/kg BW twice
at a 2-week interval to inhibit the endogenous liver cell proliferation. Two weeks after the second
injection, recipient mice were partially hepatectomized to remove 70% of the liver to create liver
injury and thus enhance the environment for the proliferation of transplanted oval cells (Figure 3-
59
1). A species-specific hAAT ELISA was performed to measure serum hAAT protein level from
the transgene expression. At the end of experiment, liver tissues were collected for
immunostaining for hAAT.
Results
Ex vivo Transduction Efficiency on Oval Cells by rAAV Vectors
To optimize the transduction efficiency of rAAV vectors on oval cells, oval cells were
infected with 4 different serotypes of rAAV vectors derived from human or nonhuman primates
or bovine rAAV vector using GFP as a reporter gene, respectively. An identical genome, AAV-
CB-GFP, were packaged into each of four different AAV serotype capsids (serotype 1, 7, 8, 9)
and bovine AAV capsid. rAAV-CB-GFP vector transduced oval cells were subjected to flow
cytometry analysis to quantify the GFP positive oval cells. As seen in Figure 3-2, rAAV1-CB-
GFP vector yielded 2.17% green fluorescent cells 7 day after infection at a dose of 104vg/cell.
rAAV7-CB-GFP and rAAV8-CB-GFP vectors yielded 2.11% and 1.63% green fluorescent cells,
respectively, under the same condition. rAAV9 and bovine rAAV vector gave less than 1% green
fluorescent cells. Consistent with our previous observation, rAAV1 vector showed the highest
transduction efficiency than other 4 rAAV vectors, although the transduction efficiency was low.
Lentiviral Vector Construction
In order to generate a lentiviral vector expressing hAAT, a lentiviral virus vector plasmid,
pTYF-linker derived from HIV-1 was used as a parental plasmid (Figure 3-3A). Human AAT
expression cassette including hAAT cDNA driven by CB promoter (Figure 3-3B) was inserted
into pTYF-linker plasmid. Briefly, the DNA fragment of hAAT cDNA and the CB promoter was
released from pCB-hAAT plasmid by enzyme digestion with BglII and BstEII. Two fragments
(2639bp and 600bp) were obtained. Since the Ploy A sequences can not be included in RNA
viral vector we have use PCR amplification to remove the poly A sequences at 3’-end of hAAT
60
cDNA. We have inserted an enzyme digestion site SpeI into the primer before the polyA site for
cloning purpose. PCR was applied to amplify the fragment between BstEII (2802) and BstEII
(3426) of pCB-hAAT. The amplified fragment was cloned into TA clone and released by
enzyme digestion with BstEII and SpeI. The BglII-BstEII (2639 bp) fragment and the PCR
amplified BstEII-SpeI fragment were cloned into pTYF-linker between BamHI and SpeI to yield
the Lenti-CB-hAAT construct (Figure 3-3C). Restriction enzyme digestion showed this construct
is correct (Figure 3-3D). This construct was packaged into lentiviral particles by Dr. Chang’s
laboratory. To evaluate the transduction efficiency of Lenti-CB-hAAT vector on oval cells, oval
cells were infected with Lenti-CB-hAAT vector at 5 MOI (multiplicity of infection). To compare
the transduction efficiency between lentiviral vector and rAAV vector, oval cells were also
infected with rAAV1-CB-hAAT and rAAV8-CB-hAAT vector at 104 MOI, respectively.
Supernatant media were assayed for transgene product (hAAT protein) using species-specific
hAAT ELISA. As shown in Figure 3-4, Lenti-CB-hAAT vector can transduce oval cells and
mediate higher transgene expression than rAAV vectors, 100-fold higher than rAAV1 and
rAAV8, while the transgene expression levels from rAAV 1 and 8 vectors are comparable.
Ex vivo Transduction and Transplantation of Oval Cells
Based on above results, we chose rAAV1-CB-hAAT and Lenti-CB-hAAT vectors for
transplantation studies. In this study, each recipient mouse received rAAV1-CB-hAAT or Lenti-
CB-hAAT vector transduced oval cells (2×106 cells/mouse). Ten weeks post transplantation,
recipients were sacrificed and the liver sections were subjected to hAAT immunostaining.
Results demonstrated that transgene hAAT was expressed in the recipient liver cells of both
groups (Figure 3-5). However, lentiviral vector resulted in fewer hAAT positive cells and much
lower transgene expression than rAAV1 vectors. Blood samples were collected every week for
evaluating the hAAT serum level. As Figure 3-6A showed, one out of five mice transplanted
61
with rAAV1-CB-hAAT vector transduced oval cells exhibited transient elevation (2,500ng/ml)
of serum hAAT at week 2 post transplantation. Other mice in both Lenti-CB-hAAT vector and
rAAV1-CB-hAAT vector groups didn’t show any significant increase in serum hAAT level
compared to saline group in which mice were transplanted with untransduced oval cells. Immune
responses to the transgene product (hAAT protein) were examined. Serum anti-hAAT IgG level
increased as the serum hAAT concentration increased (Figure 3-6B).
Discussion
Results from these studies confirmed rAAV vector can be used for genetic modification of
oval cells. Several rAAV vectors including serotype 1, 7, 8, 9 and bovine AAV were tested for
transduction efficiency on oval cells using flow cytometry for quantification. Although the
transduction efficiencies for all vectors were low, rAAV1 is the best among the vectors tested.
Using GFP as report gene, flow cytometry demonstrated less than 3% oval cells were green cells
one week after infection. The transduction efficiency estimated by GFP positive cells might be
underestimated for several possible reasons. First, rAAV vector is single stranded DNA vector
and the transgene expression requires the conversion of ssDNA to dsDNA which depends on the
host cell DNA replication machinery. Host cell stage may also have effect on the transgene
expression. Some cells demonstrated early transgene expression while others were on the late
stage. Usually it takes 4-6 weeks for rAAV transgene expression to reach maximum in vivo.
However, in vitro propagation of stem/progenitor cells will dilute out the rAAV vector.
Therefore, the evaluation of oval cell transduction was relative and helpful for selection of better
vector. This approach can not be suitable for absolute quantification of transduction, which may
require quantification of vector DNA in the cells. Nevertheless, our results consists previous
observation and showed rAAV1 is the best one that mediated the highest transgene expression on
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oval cells among different AAV serotypes (serotype 1-5 and 7-9), and bovine AAV. Hence
rAAV1-CB-hAAT vectors were used for in vivo transplantation study.
We observed transgene expression in the recipient liver after transplanting rAAV1-CB-
hAAT vector transduced oval cells. We also noticed the large variation among the animal on
transgene expression. In contrast to previous study, current study only observed transient
transgene product (hAAT protein) in the serum. One possible explanation is that 5 times lower
MOI was applied in this study. In addition, ELISA demonstrated a significant increase in serum
anti-hAAT IgG level, as we observed in the study of rAAV1- mediated hAAT gene deliver to
skeletal muscle.76 No antibody response was seen when there is a lack of detectable circulating
hAAT. These results suggest that anti-hAAT antibody might target and neutralize the transgene
product (hAAT), and thus limited the concentration of circulating hAAT. For future studies,
SCID mice may be used as recipient to rule out this problem.
In order to enhance the transgene expression, lentiviral vector was constructed and used for
hAAT gene delivery into the oval cells. We showed that Lenti-CB-hAAT vector infected oval
cells efficiently and mediated efficient transgene expression in cell culture condition. However,
after transplantation of transduced cells, hAAT expression in the recipient liver was very low or
nearly undetectable. There are two possible explanations. Studies conducted by Brown and
colleagues showed that in vivo administration of lentiviral vector to mice triggered a type I
interferon response.248 This innate immune response in turn triggered an adaptive response
against transgene product and also promoted vector clearance. In addition, lentiviral vector
integrates into the host genome randomly. It has been demonstrated that transgene expression is
subjected to negative influence of chromosomal sequence flanking the integration sites and often
leads to transcriptional silencing.131, 249, 250 The interaction between the cis-acting elements of
63
provirus and trans-factors of stem cells results in epigenetic modifications including DNA
methylation and chromatin structure modulation, which may contributes to the lentiviral
transgene silencing.249, 251, 252 Transcriptional silencing is most pronounced in stem cell. The
trans-factors scan for foreign DNA and establish silencing in stem cells and maintain silencing in
their progeny.252 Strategies for overcoming these limitations are under the development including
addition of chromatin insulator element to protect the transgene from negative position effect or
deletion/mutation of the retrovirus silencer elements.251, 253-255
Although our studies have clearly demonstrate the feasibility of stem cell mediated hAAT
gene delivery using rAAV vector and oval cells, isolation of oval cell for autologous
transplantation in human is not practical. Therefore, next studies will focus on finding new stem
cell resources for liver directed hAAT gene delivery.
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 transplantation. Liver oval cells were isolated from male GFP transgenic C57BL/6 mice treated with a diet containing 0.1% DDC for 4 weeks. Magnetic cell sorting (MACS) system was applied to isolate Sca l+ cell population from the nonparenchymal cell compartment from 2-step liver perfusion. The newly purified Sca l+ oval cells were infected with rAAV1-CB-hAAT vector and Lenti-CB-hAAT vector for 2 hours, respectively, washed and transplanted into the recipient liver by intrasplenic injection. Serum hAAT levels were monitor by ELISA. Liver tissues were subjected to hAAT specific immunostaining.
65
Figure 3-2. Flow cytometric quantification of green fluorescent oval cells after transduction of rAAV-CB-GFP vectors.
66
Figure 3-3. Construct of Lenti-CB-hAAT vectors. (A) Lentiviral vector backbone. pTYF-Linker is derived from an LTR-modified recombinant HIV-1 plasmid. (B) hAAT expression cassette. hAAT is driven by CB promoter. (C) Restriction map of Lenti-CB-hAAT plasmid. (D) Gel electrophoresis results. H: HindIII; SB: SpeI & BamHI; S: SpeI; B: BamHI.
67
Figure 3-4. Ex vivo transduction of oval cells. Mouse oval cells were grown in the 24-well plate (1×104 cells/well; n=3) and were infected with the Lenti-CB-hAAT vector at 5 MOI, rAAV1-CB-hAAT and rAAV8-CB-hAAT vectors at 1×104 MOI, respectively. The accumulative hAAT in the culture medium was measured by ELISA. Open triangle, lentiviral vector; Closed circle, rAAV1 vector; Open square, rAAV8 vector.
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.
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 2hr, respectively, and transplanted into hepatectomized mouse liver ( n=5 each group) by intrasplenic injection. Serum samples were collected very week for detection of hAAT. (B) Serum anti-hAAT IgG levels were estimated by ELISA. Open circle: Lenti-CB-hAAT; Open square: rAAV1-CB-hAAT; Cross, Saline; Open triangle: one mouse in rAAV1-CB-hAAT group with transient increase in serum hAAT level at week 2 post transplantation. Dash is lower limit of quantification (LLOQ).
70
CHAPTER 4 EX VIVO TRANSDUCTION AND TRANSPLANTATION OF BONE MARROW CELLS FOR
LIVER GENE DELIVERY OF ALPHA 1-ANTITRYPSIN
Summary
Adult stem cell-based gene therapy holds several unique advantages including avoidance
of germline or other unwanted cell transduction. We have previously showed that liver
progenitor (oval) cells can be used as a platform for liver gene delivery of human alpha 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 vitro studies showed that bone marrow cells can be transduced
by lentiviral vector (Lenti-CB-hAAT) and recombinant adeno-associated viral vectors (rAAV1-
CB-hAAT, and rAAV8-CB-hAAT). Transplantation studies showed that transplanted bone
marrow cells homed into liver, differentiated into hepatocytes and expressed hAAT in the liver.
Importantly, we showed that transplantation of rAAV8-CB-hAAT vector transduced bone
marrow cells resulted in long-term and sustained levels of hAAT in the systemic circulation of
recipient mice. These results demonstrated that rAAV vector mediated, bone marrow cell-based
liver gene therapy is feasible for the treatment of AAT deficiency and implies a novel therapy for
the treatment of liver diseases.
Introduction
Alpha 1-antitrypsin deficiency (AATD) is a genetic defect caused mostly by a single base
substitution in the alpha 1-antitrypsin (AAT) gene which encodes a 52kDa glycoprotein.256 This
mutation results in accumulation of polymerization of mutant AAT protein in the hepatocytes
where AAT is mainly synthesized and secreted into the circulation, and consequently leads to a
reduced level of AAT in the serum.257 As a serine protease inhibitor, the primary function of
AAT is to protect delicate tissue such as pulmonary interstitial elastin against the excessive
71
proteolytic damage of neutrophil elastase (NE). Deficiency of AAT in the serum could cause
alveolar elastin exposure to NE and lead to an increasing risk of developing early onset
pulmonary emphysema by ages 35 to 50 years if the AAT serum level is less than 11µM
(approximately 800ug/ml).247 Aggregated mutant AAT in the endoplasmic reticulum of
hepatocytes would result in the liver disease such as neonatal jaundice and hepatic cirrhosis
which take places in one portion of patients homozygous for PI*Z mutation.247 For AAT
deficiency-associated lung disease, restoring anti-NE protection in the lung has been achieved by
boosting serum AAT level via weekly intravenous infusion of human plasma AAT.258 Strategies
of overexpression of wild-type AAT gene to correct the deficiency of AAT by gene transfer to
muscle are being investigated in the phase I clinical trial using recombinant adeno-associated
virus vector (rAAV) serotype 1 & 2.106 For AAT deficiency- associated liver disease, no
effective therapy is available except liver organ transplantation which is limited by the shortage
of donor organ and immune rejection.
Adult stem cells offer a platform for ex vivo genetic manipulation followed by autologous
transplantation, which will overcome many limitations, including immune rejection of allogeneic
cells and ethical issue of embryonic stem cells. Non-specific targeting, such as germline
transduction, is one of the major concerns of conventional gene therapy, direct infusion of gene
into a patient. Using stem cell as a mean for delivering gene into patients could minimize the
unwanted cell transduction. More importantly, stem cell, a regenerative medicine, can self-renew
and continue replenishing the aged or damaged tissue cells, and thus stem cell-based gene
therapy may reduce or eliminate the need for repeated administration of the gene therapy. Adult
stem cell gene therapy which replaces the patients’ disease-causing gene with the healthy
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 progenitor cells.145, 162, 164 Sex-
mismatched BM transplantation in lethally irradiated DPPIV¯ rat treated with 2-AAF/CCl4 has
firstly demonstrated cells in the bone marrow are capable of repopulating the injured liver.145
Both cell fusion with host hepatocytes and hepatic transdifferentiation of BM cells have been
proposed as the underlying principle.206, 207
AAV vector, a nonpathogenic vector, is able to transduce a broad range of tissues and cells
and mediate long-term transgene expression. However, the transduction efficacy of rAAV vector
in HSCs is not conclusive. Whereas some researches showed that HSCs can be successfully
transduced by rAAV vector and the rAAV-transduced HSCs maintain their multipotential
differentiation, long-term persistence and self-renewal capacity, others have reported the HSCs
are impervious to rAAV vector.107, 108 Unlike HSCs, MSCs can be efficiently transduced by
rAAV vector. rAAV vector have been applied for gene targeting in MSCs.110 In the present
study, we tested the feasibility of BM cell mediated liver gene delivery of hAAT in mouse
model.
Animal Experimental Design
The recipients, 4-week old female C57BL/6, were IP injected twice (2 weeks interval) with
MCT at 50mg/kg BW and received PHx before transplantation. BM cells were isolated from the
femurs and tibias of 6 to 8-week-old male C57BL/6 GFP transgenic mice. The newly isolated
BM cells were infected with Lenti-CB-hAAT vector at 1×102 MOI, rAAV1-CB-hAAT and
73
rAAV8-CB-hAAT vector at 1×104 MOI, respectively, for 2 hours. After transduction, BM cells
were washed 3 times with PBS and then resuspended in saline solution at an approximate
concentration of 5×106 cells/100ul. Transduced BM cells were transplanted into the recipient
liver (5×106 cells/mouse) by intrasplenic injection (n=4) or portal vein injection (n=5). Serum
samples were collected every week post transplantation. Serum hAAT levels were monitored by
ELISA. Recipient mice were sacrificed at 8 or 14 weeks post transplantation, and liver tissues
were harvested for immunostaining to evaluate the engraftment efficiency and transgene
expression (Figure 4-1).
Results
Bone Marrow Cells Transduction
In order to test the transduction efficiency of bone marrow cells, total bone marrow cells
were isolated and infected with Lenti-CB-hAAT, rAAV1-CB-hAAT and rAAV8-CB-hAAT
vectors. As shown in Figure 4-2, all vectors can transduce BM cells, but with different
efficiency. Lenti-CB-hAAT infection resulted in the highest levels of hAAT in culture medium.
Although hAAT levels in rAAV vector infected cells are much lower than that in lentiviral
vector infected cells, they are clearly detectable. Relatively, rAAV1-CB-hAAT mediated higher
levels of hAAT than rAAV8-CB-AAT. These results suggest that both lentiviral vector and
rAAV vectors can be useful in bone marrow cell transplantation studies.
Liver Transplantation of ex vivo Transduced Bone Marrow Cells
Next, we tested the feasibility of bone marrow cell transplantation for liver gene delivery
of alpha 1-antitrypsin. As described in Figure 4-1, male GFP transgenic mice were used as donor
animals and female C57BL/6 mice were used as recipients. The recipients were treated with
monocrotaline (MCT) to inhibit endogenous hepatocyte proliferation. The recipients also
received partial hepatectomy (PHx) before transplantation to create liver injury, thus enhancing
74
the environment for the proliferation and differentiation of transplanted bone marrow cells. In
this experiment, freshly isolated total bone marrow cells (donor cells) were infected with Lenti-
CB-hAAT (MOI=100), rAAV1-CB-hAAT (MOI=1x104) and rAAV8-CB-hAAT (MOI=1x104)
vectors, respectively. After thorough washes, 5x106 cells were transplanted into recipient liver
through portal vein injection. As shown in Figure 4-3, rAAV8-CB-hAAT vector mediated more
hAAT positive cells in the recipient liver, while Lenti-CB-hAAT and rAAV1 vector mediated
few hAAT positive cells. To confirm that the hAAT positive cells were derived from donors, we
had performed co-immunostaining experiments. As shown in Figure 4-4, hAAT positive cells
from rAAV8-CB-hAAT vector treatment group were also GFP positive.
Bone Marrow Cell Transplantation Resulted in Sustained Levels of hAAT in Recipient Circulation
In order to further enhance transgene (hAAT) expression, we have performed an additional
experiment with a modified procedure.245 In this experiment, we transplanted 2x107 rAAV8-CB-
hAAT infected bone marrow cells (from male C57BL/6) into the recipients through intrasplenic
injection. As expected, Y-chromosome positive cells were detected in the recipient liver
demonstrating that donor cell could migrate and integrate into recipient liver (Figure 4-5).
Immunostaining studies showed that most of hAAT positive donor cells were also positive for
mouse albumin in the liver indicating that majority of donor bone marrow cells in liver
transdifferentiated into hepatocytes (Figure 4-6, black arrow). To test the possibility that some
bone marrow cells could home to other organs and express transgene, we had performed Y-FISH
and immunostaining in spleen, bone and lung. Both Y-FISH and AAT-immunostaining showed
some donor cells retained in spleen (Figure 4-7A and 4-7B). AAT-immunostaining also showed
some AAT-positive cells in lung and bone marrow indicating that intrasplenic injection of bone
marrow cells also resulted in multi-organ homing of these cells (Figure 4-7C and 4-7D, black
75
arrow). Importantly, long-term and sustained serum levels of hAAT were obtained in this
experiment (Figure 4-8). These results imply that transplantation of rAAV8-transduced bone
marrow cells represents a novel therapy for AAT deficiency.
Discussion
Adult stem cells offer a platform for ex vivo genetic manipulation followed by autologous
transplantation. Adult stem cell-mediated gene delivery may overcome many limitations,
including immune rejection of allogeneic cells and ethical issue of embryonic stem cells, the
shortage of donor organs, and non-specific targeting (such as germline transduction). In addition,
adult stem cells-mediated gene therapy can serve as a regenerative medicine to replace diseased
cells with patient’s stem cells carrying healthy gene(s). We have previously showed that liver
stem (or progenitor) cell mediated liver gene delivery of hAAT was feasible in mouse model.245
However, liver stem cells can not be used in humans for autologous transplantation. Considering
clinical practice, we investigated the possibility of transplanting genetically modified BM cells.
In the present study, we showed that rAAV8-CB-hAAT vector transduced BM cells
differentiated into hepatocytes and mediated sustained serum levels of hAAT in mouse model.
These results imply a novel therapy the treatment of alpha 1-antitrypsin deficiency in humans.
In the present study, we showed that Lenti-CB-hAAT vector transduced BM cells
efficiently in vitro. Interestingly, transplantation of these transduced cells resulted in
undetectable levels of hAAT in the circulation, although some hAAT positive cells were detected
in the recipient mouse liver. These results were consistent with the previous oval cell studies.
The possible mechanisms discussed in Chapter 3 may apply here as well.
BM cells can be transduced by both rAAV1 and rAAV8 vectors in vitro. However, much
higher levels of hAAT were detected in liver and serum from recipients received rAAV8-CB-
hAAT infected BM cells than that from recipients received rAAV1-CB-hAAT infected BM
76
cells. It is possible that the transdifferentiation of BM cells into hepatocytes provide favorable
cellular environment for the intracellular process of rAAV8 vector including cytoplasmic
trafficking, uncoating, and nuclear entry. Recent studies have shown that mutations of capsid
proteins can affect rAAV2 vector trafficking and enhance transgene expression, and might
support above hypothesis.59 Future studies will investigate the effect of stem cell
transdifferentiation on rAAV vector intracellular processing.
BM cells contain HSCs and MSCs, and both have been shown to possess hepatic-
differentiation potential.162, 195, 198 Furthermore, considering the possible contribution of cell to
cell interaction to stem cell proliferation and differentiation, the present study employed total
bone marrow cells. After intrasplenic injection, donor cells were found not only in the liver, but
in spleen, lung, and bone marrow. It was expected that some donor bone marrow cells were
trapped in the injection site, spleen, while some cells home back to the bone. The BM migrating
or homing to lung might due to the pulmonary toxicity induced by MCT, a pyrollizidine alkaloid
(PA) plant toxin. MCT is bioactivated by cytochromes P450 in hepatocytes to its active
compound monocrotaline pyrrole (MCTP) that produces both hepatic and pulmonary toxicity.259
The results of the present study suggested that BM cell-based gene therapy approach is a
promising therapy for genetic diseases. However, further improvement on transgene expression
in the donor cells is required to achieve therapeutical application. This issue could be addressed
mainly from two aspects, transduction efficiency by viral vectors and the
transplantation/engraftment efficiency of adult stem cells. Rapid advances in gene delivery
vector provide future gene therapy with lots of options. Self-complementary AAV vectors
circumvent rate-limiting second-strand synthesis in single-stranded AAV vector genome and
thus facilitate robust and highly efficient transduction.87 Site-specific integration AAV vector can
77
establish long-term and persistent gene expression. Integration to AAVS1 locus could resist to
transgene silencing, a major obstacle of integration viral vector such as retroviral vector.102 The
efficiency of differentiation of BM-derived MSCs can be improved by modifying the culture
conditions such as adding growth factors or cytokines, or by delivering an expression cassette to
regulate hepatic differentiation.195, 198, 260 Pre-conditioned MSCs demonstrated higher liver
engraftment potential.
Here we showed that BM cells can be transduced by rAAV8 vector and that transplantation
of these cells resulted in hepatic differentiation and transgene expression in the liver and
detectable levels of transgene product (hAAT protein) in the serum. Detailed studies to elucidate
the mechanism underlying interaction between viral vectors and adult stem cell, stem cell
regulation on expression of foreign gene, migration and homing of stem cell will enhance the use
of BM cell-based gene therapy for the treatment of AAT deficiency.
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Figure 4-1. Experimental outline of BM cells study. The recipients (female C57BL/6) were IP injected twice (2 weeks interval) with 50mg/kg of monocrotaline (MCT) and received partial hepatectomy (PHx) to remove 70% of liver mass before transplantation. BM cells were isolated from the femurs and tibias of male C57BL/6 mice. The newly purified BM cells were infected with Lenti-CB-hAAT, rAAV1-CB-hAAT, rAAV8-CB-hAAT vector, respectively, for 2 hours, washed, and transplanted into the recipient liver by portal vein injection or intrasplenic injection. Serum hAAT levels were monitor by human AAT specific ELISA. Liver repopulation was measured by immunostaining.
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Figure 4-2. Ex vivo transduction of BM cells. Mouse Bone marrow cells were seeded in 24-well (1×104 cells/well; n=3) and were infected with the Lenti-CB-hAAT vector at 100 moi, rAAV1-CB-hAAT, rAAV8-CB-hAAT at 104 moi, and PBS, respectively. The accumulative hAAT in the culture medium was measured by ELISA. Circle, 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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Figure 4-3. Detection of expression of human alpha1-antitrypsin (hAAT) in recipient liver after transplantation of viral vector infected BM cells by immunostaining. (A) Liver section from C57BL/6 mouse transplanted with Lenti-CB-hAAT infected BM cells (Brown). (B) Image A view at larger magnification. (C) Liver section from C57BL/6 mouse transplanted with rAA1-CB-hAAT infected BM cells. (D) Image C view at larger magnification. (E) Liver section from C57BL/6 mouse transplanted with rAAV8-CB-hAAT infected BM cells. (F) Image E view at larger magnification. (G) Human liver section served as positive control. (H) Liver section from untransplanted C57BL/6 mouse served as negative control.
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Figure 4-4. Detection of transgene expression from the engrafted donor BM cells by fluorescence double immunostaining for human alpha 1-antitypsin (hAAT) and green fluorescent protein (GFP). (A) Liver section from C57BL/6 mouse transplanted with rAAV8-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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Figure 4-5. Detection of donor cells in recipient liver after BM cell transplantation by fluorescence in situ hybridizations (FISH) for Y-chromosome. (A, B, C) Female mice treated with MCT/PHx and BMTx from male mouse. x, X chromosome; Y, Y chromosome.
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Figure 4-6. Detection of coexpression of human alpha 1-antitypsin (hAAT) and mouse albumin by immunostaining. (A, C) Liver section from C57BL/6 mouse transplanted with rAAV8-CB-hAAT infected BM cells stained for hAAT (brown). (B,D) Liver section adjacent to the section in A and C, respectively, stained for albumin (red). Black arrow point to both AAT positive and albumin positive cells. Asterisks: location indicator. Representative slides were viewed at ×20 magnification.
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Figure 4-7. Multi-organ homing of transplanted BM cells. Tissue sections were from female C57BL/6 mouse at 8 weeks after transplantation with rAAV8-CB-GFP vector infected male BM cells. (A) Spleen section subjected to FISH for detecting Y-chromosome. (B) Spleen section stained for GFP (brown). (C) Bone section stained for GFP. (D) Lung section stained for GFP. Black arrowheads indicate the observed GFP staining.
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Figure 4-8. Detection of expression of human alpha1-antitrypsin (hAAT) in the recipient serum. BM cells from C57BL/6 mice were infected with ssAAV8-CB-hAAT vector at 1×104 particles/cells for 2 h and transplanted into liver of partially hepatectomized C57BL/6 recipient (2 x107 cells/mouse; n=4) by intrasplenic injection. The transgene expression was monitored by measuring the serum level of hAAT. Square is the serum from the treatment group; Dash is lower limit of quantification (LLOQ). The serum level of hAAT from untransplanted C57BL/6 mouse (negative control) was below the LLOQ.
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CHAPTER 5 ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELL-BASED LIVER GENE
DELIVERY
Summary
Adipose tissue represents an accessible, abundant, and replenishable source of adult stem
cells for potential application in regenerative medicine. Adipose tissue-derived mesenchymal
stem cells (AT-MSCs) resemble bone marrow-derived mesenchymal stem cells (BM-MSCs)
regarding morphology, immune phenotype and multiple differentiation capability, while possess
the advantage of less invasive procurement and obtainability in large quantity. In light of recent
observation of hepatic differentiation of AT-MSCs, our study investigated the feasibility of 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 transduced by recombinant adeno-
associated viral vector serotype 1(rAAV1-CB-hAAT). After transplanting to MCT/PHx injured
liver, ex vivo transduced AT-MSCs displayed sustained transgene expression and secreted
transgene product, human alpha 1-antitrypisn (hAAT) into circulating system resulting in a
serum hAAT level of 100-200ng/ml. Immunostaining for hAAT on recipient liver section
revealed that about 5-10% recipient liver was repopulated from approximately 1.6 ×106 ex vivo
genetically modified AT-MSCs, 8 weeks post transplantation. More importantly, AT-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, contributed to liver regeneration, and served as platform for transgene expression.
AT-MSC-based gene therapy presents a novel approach for the treatment of human genetic
diseases, such as AAT deficiency.
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Introduction
Alpha 1-antitrypsin (AAT) deficiency is a genetic disorder resulting from a single gene
mutation on AAT coding gene, which results in a reduction of serum levels of AAT and
accumulation of AAT protein in hepatocytes. Consequently this mutation causes an increased
risk of developing early onset pulmonary emphysema and severe forms of liver disease,
including neonatal jaundice, cirrhosis, and hepatitis.1, 2 Protein replacement therapy consisting of
weekly repeated intravenous infusion of human AAT (hAAT) is the only available treatment for
AAT deficiency-associated lung disease so far, while this therapy is expensive, not a cure, and
temporary effect. For AAT deficiency-associated liver disease, no effective therapy is available
except liver organ transplantation which is hampered by the shortage of donor organ and immune
rejection. To circumvent this dilemma, we propose to replace dysfunctional hepatocytes with ex
vivo genetically modified adult stem cells which carry the correct AAT gene.
An urgent need for an adequate supply of hepatocytes for liver repopulation drives
researchers to investigate in generating hepatocytes from extrahepatic adult stem cells, such as
MSCs. MSCs are a heterogeneous population of plastic-adherent, spindle-shaped and fibroblast-
like cells which can be extensively expanded in vitro whilst retaining their multi-lineage
differentiation potential such as osteogenesis, chondrogenesis, adipogenesis.189, 190 In addition to
differentiation into its native derivatives, mesenchymal tissues, MSCs also have the potential to
differentiate into hepatocytes in vitro and in vivo.195, 198 MSCs have been traditionally isolated
from bone marrow aspirates with a yield of approximately 1 MSC per 105 BM nucleated cells.190
To be clinical usefulness, such low cell number necessitate ex vivo expansion to obtain clinically
significant cell numbers, which is time consuming and risk of cell contamination. Furthermore,
differentiation potential and maximum life span of MSCs from BM decline with increasing age.
217-219 Fortunately, MSCs can also be isolated from adipose tissue.221, 261 There is little to no
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difference between BM-MSCs and AT-MSCs regarding the morphology, immune phenotype,
yield of adherent stromal cells, growth kinetics, cell senescence, multilineage differentiation
capacity or transduction efficiency.197, 213 More importantly, the frequency of colony-forming
unit-fibroblasts (CFU-F) in adipose tissue is hundred-fold higher than that of bone marrow.197
From practical standpoint, adipose tissue may represent an idea autologous stem cell source of
repeatable access, replenishment, easy isolation, and minimal patient discomfort.
Adeno-associated virus (AAV) is a linear single-stranded DNA parvovirus with a genome
of 4.7 kb and a non-enveloped capsid of approximately 22 nm in diameter.38 To date, total 12
AAV serotypes and over 100 AAV variants have been discovered from human/nonhuman
primate tissues. AAV serotypes display distinct and broad cell and tissue affinities such as
muscle, liver, lung, and central nervous system.262 Not any human diseases have been shown to
be associated with AAV. The lack of pathogenicity, low risk of insertional mutagenesis, many
available serotypes, and broad tissue tropisms have make rAAV vector rapidly gain popularity in
gene therapy application. Since 1995 AAV2-based vector was first administrated to a human
subject for treating cystic fibrosis,263 over 40 clinical trials have now been approved involving 14
diseases so far.106 These studies indicate that in vivo gene transfer is feasible and relatively safe,
but also suggest that the transduction efficiency of AAV2 vectors fall short of requirement for
adequate and organ-specific transgene expression. As a result, ongoing research efforts are
focused on developing new AAV vector by modifying both AAV genome and capsid protein.
For example, self-complementary double-stranded AAV (dsAAV) vectors generated by mutating
one of the AAV ITRs exhibit faster onset of gene expression and higher transduction efficiency
than single-stranded AAV (ssAAV) vectors in muscle, liver and brain.86, 87 The underlying
mechanism could be dsAAV vectors bypass the rate-limiting step that host cell mediates
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synthesis of dsDNA from the ssDNA.86 Other efforts have focused on engineering capsid protein
such as transcapsidation, adsorption modification, mosaic capsid, and chimeric capsid.65 For
instance, AAV2 ITR has been cross-packaged into AAV1 capsid and tested in clinical trial for
muscle–directed gene therapy for AATD because rAAV1 vectors have shown hundred-fold more
potency for murine muscle transduction than rAAV2 vectors.37, 76 Furthermore, rAAV vectors
have been demonstrated to be capable of transducing MSCs efficiently and the transduced MSCs
retained multipotential activities.110
The combination of rAAV-mediated gene delivery with stem cell therapy is the future
direction. The capability of self-renewal and differentiation of stem cells make them to be the
promising target of virus vector for long-term gene correction. By putting viral vectors in the
stem cell, we can limit the undesired side effect resulting from the nonspecific targeting by
systemic delivery of rAAV vectors. Here, we demonstrated that MSCs from mouse peritoneal
adipose tissue can be genetically modified by rAAV vector and transplanted into liver
parenchyma. Engrafted AT-MSCs was able to mediate long-term transgene expression.
Experimental Design
In vivo Transduction by ssAAV and dsAAV Vectors
Female C57BL/6 mice (8-week old) were injected with ssAAV and dsAAV vectors to
compare the transduction efficiency. Four rAAV vectors were selected, ssAAV1-CB-hAAT,
dsAAV1-CMV-hAAT, ssAAV8-CB-hAAT, and dsAAV8-DHBV-hAAT. Two groups of mice
(n=5, each) received rAAV1 (ssAAV and dsAAV) by percutaneous injection into the quadriceps
femoris muscles of both hind limbs with 2×1010 particles per mouse in 50ul saline, respectively.
The other two groups of mice (n=5, each) received rAAV8 (ssAAV and dsAAV) by portal vein
injection with 2×1010 particles per mouse in 50ul saline. Serum samples were taken from tail vein
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every week post injection and subjected to hAAT specific ELISA to evaluate the transgene,
hAAT, expression.
Ex vivo Transduction and Transplantation of AT-MSCs
The recipients, 4-week old female C57BL/6, were IP injected twice (2 weeks interval) with
MCT at 50mg/kg BW and received PHx before transplantation. AT-MSCs were isolated from
the peritoneal adipose tissue of 6 to 8-week-old male C57BL/6 mice. The newly isolated AT-
MSCs were infected with ssAAV1-CB-hAAT vector at 5×104 MOI for 2 hours. After
transduction, AT-MSCs were washed 3 times with PBS and then resuspended in saline solution
at an approximate concentration of 1.6×106 cells/100ul. Transduced AT-MSCs were transplanted
into the recipient liver (1.6×106 cells/mouse, n=5) by intrasplenic injection. Serum samples were
collected every week post transplantation. Serum hAAT levels were monitored by ELISA. 8
weeks post transplantation, liver tissue of recipient mice were harvested for immunostaining
(Figure 5-1).
Results
Isolation and Characterization of AT-MSCs
Mouse AT-MSCs were isolated from peritoneal adipose tissue of male C57BL/6 mice as
described in Chapter 2. These cells were characterized by immunofluorescence and multiple
differentiation potential upon exposure to adipogenic and osteogenic induction medium.
Immunofluorescence staining revealed that the cells isolated from the mouse peritoneal adipose
tissue expressed stromal-associated marker CD44, CD90 and CD105 but didn’t express either
hematopoietic markers CD34 and CD45 or endothelial marker CD31. The expression of CD133
was low (Figure 5-2A). The cell surface phenotypes were consistent with those reported in the
literature for adipose tissue derived stem cells.222 Multiple differentiation potential of AT-MSCs
was demonstrated by induced differentiation into adipocytes and osteocytes. Two weeks after
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exposure to adipogenic induction medium, intracellular lipid droplets were observed within the
adipogenic-differentiated AT-MSCs using Oil Red O staining. Osteogenic differentiation
resulted in extracellular calcium phosphate precipitates as revealed by Alizarin Red S staining
(Figure 5-2B).
Optimization of rAAV Vecotors
AAV is a single stranded DNA virus. After infection, the viral DNA undergoes second
stranded DNA synthesis using host cellular enzymes. Therefore, transgene expression from
conventional single stranded rAAV vector depends on the second-stranded DNA synthesis.
Recently double-stranded AAV vectors (dsAAV), or self-complementary AAV vectors(scAAV)
have been developed by deleting the D-sequence (the packaging sequence) and the adjacent
terminal resolution site (trs) of one of the ITRs. It has been shown that dsAAV can avoid second-
stranded synthesis in the host cells thus mediated quicker and higher levels of transgene
expression. In order to achieve optimal levels of hAAT expression in AT-MSCs, we have
generated two dsAAV vectors, dsAAV-DHBV-hAAT and dsAAV-CMV-hAAT. Due to the
limitation of the packaging capacity of dsAAV (2.4 kb, half of the full packaging capacity of
ssAAV vector of 4.7 kb), two smaller promoters were used. Duck hepatitis B virus (DHBV)
promoter has been shown as an active liver specific promoter.264 CMV promoter was also used
since it is active in most of the stem cells and muscle cells. The dsAAV-CMV-hAAT vector
plasmid was packaged into rAAV1 vector for muscle gene delivery. As shown in Figure 5-3A,
dsAAV1-CMV-hAAT mediated detectable levels of hAAT expression. However, the levels were
lower than that from matched dose of ssAAV1-CB-hAAT vector. Similarly, the dsAAV-DHBV-
hAAT vector plasmid was packaged into rAAV8 vector for liver gene delivery. As shown in
Figure 5-3B, dsAAV8-DHBV-hAAT mediated sustained levels of hAAT, but the levels were
much lower than that from ssAAV8-CB-hAAT vector. These results suggested that advantage of
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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 vectors were used to infect AT-MSCs. As shown in Figure 5-4A, ssAAV1-CB-hAAT
vector mediated the highest transgene expression than the other three rAAV vectors, dsAAV1-
CMV-hAAT, ssAAV8-CB-hAAT, and dsAAV8-DHBV-hAAT, as indicated by more than 10-
fold increase in hAAT serum level at day 9 post transduction. It was interesting that rAAV1
mediated more than 25-fold higher levels of hAAT than rAAV8 in AT-MSCs. Furthermore,
double infection of AT-MSCs using ssAAV1-CB-hAAT vector at 12hr interval could further
increase the transgene expression (Figure 5-4B). Together, above results clearly demonstrated
the ssAAV1-CB-hAAT vector was the best vector among those tested and displayed two
advantages in vector transduction and transcription of transgene. Therefore, we decided to use
ssAAV1-CB-hAAT vector for the following studies.
Liver Transplantation of ex vivo Transduced AT-MSCs
We hypothesis that ex vivo transduced AT-MSCs with rAAV1-CB-hAAT could serve as a
platform for liver expression of hAAT after autologous transplantation. To test this hypothesis,
AT-MSCs (1.6 ×106) from male mice were infected with ssAAV1-CB-hAAT (MOI=5x104) and
were transplanted into the liver of MCT-treated and partial-hepatectomized female C57BL/6
recipients (Figure 5-1). MCT, a pyrrolizidine alkaloid, is metabolized in the liver to its active
derivatives within a few hours or days but able to induce persistent inhibition effect on recipient
hepatocyte proliferation and thus provide subsequently transplanted cells with a proliferative
advantage over endogenous hepatocytes. Partial hepatectomy was applied to create a liver
damage model for enhancing the engraftment and proliferation of transplanted AT-MSCs.
Recipient organs were harvested 8 week post transplantation and subjected to hAAT
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immunostaining. Immunostaining for hAAT revealed that 5-10% of total hepatocytes displayed
hAAT transgene expression (Figure 5-5). Those hAAT positive hepatocytes indicated that
rAAV- transduced AT-MSCs were able to migrate into liver from the injection site of spleen,
engraft into the recipient liver parenchyma, contribute to liver repopulation, and give rise to
transgene expression. More importantly, those AAT positive cells were morphologically similar
to hepatocyte. Y-chromosome fluorescent in situ hybridization (Y-FISH) further confirmed the
presence of male donor cells in the female recipient liver (Figure 5-6). To test the hypothesis that
AT-MSCs can transdifferentiate into hepatocytes after liver transplantation, serial sections of
recipient liver tissue were subjected to mouse albumin and human AAT immunostaining,
respectively. As shown in Figure 5-7, most of hAAT positive cells were also positive to mouse
albumin. These results indicated that adipose tissue-derived MSCs were able to differentiate into
functional hepatocytes with capability of producing albumin. Using GFP as reporter gene, donor
cells were also detected in spleen, lung and bone marrow after intrasplenic injection by using
immunostaining for GFP (Figure 5-8).
In order to quantify transgene product generated and secreted from engrafted 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
demonstrated that AT-MSCs can be used as platform for liver-directed hAAT gene delivery.
Discussion
AT-MSCs represent an excellent cell source for regenerative medicine. However, the use
of AT-MSCs for liver regeneration and gene delivery remain elusive. In this study, we isolated
and characterized mouse AT-MSCs. We showed that these AT-MSCs can be efficiently
transduced by ssAAV1-CB-hAAT vector. Transplantation of rAAV transduced AT-MSCs
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resulted in hepatic differentiation and sustained long-term transgene expression in the liver and
the serum of the recipients. Results from this study demonstrated that it is feasible to use AT-
MSCs as a cell vehicle for liver gene delivery and implied a novel therapy for the treatment of
liver diseases.
AAT is a major serum protective protein. It is generally accepted that patients with serum
AAT levels below 11µM or 800µg/ml may develop emphysema. Therefore, the serum level of
hAAT obtained from AT-MSCs transplantation in this study remain further improvement to be
therapeutic (approximately 500-800ug/ml)12. Several possible approaches may be employed to
enhance hAAT expression levels. Banas and his colleagues demonstrated that CD105+ fraction
of AT-MSCs exhibited high hepatic differentiation ability.232 Therefore, enrichment of stem cell
population by isolating CD105+ AT-MSCs may increase the total number of hepatocytes derived
from donor AT-MSCs and thus enhance hAAT levels in the recipient serum. Similarly, other cell
markers may also be used to enrich stem cell population, such as p75 neurotrophin receptor
(p75NTR), which has also been used to isolate and collect putative multipotent stem cell from
mouse adipose tissue-derived stromal vascular fraction culture cells (ADSVF cells).265 Secondly,
hepatic-differentiation potential of AT-MSCs can be further improved by in vitro
preconditioning toward hepatocyte-like cells such as incubating MSCs with specific growth
factors e.g., hepatocyte growth factor (HGF), epidermal growth factor (EGF) or fibroblast
growth factor (FGF).226, 232 In addition, improvement of transduction efficiency of AT-MSC by
further optimize rAAV vectors may also enhance hAAT levels in the receipt serum. In the
present study, we showed rAAV1 was more efficient than rAAV8. Other serotypes of AAV
vectors and recently developed mutant AAV vectors might mediate higher transduction
efficiency in AT-MSCs. In contrast to previous study 87, our study showed dsAAV vectors didn’t
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demonstrate superior transduction efficiency to ssAAV vector. This inconsistency to the well-
documented characteristics of dsAAV might reflect the inferior promoter activity of DHBV and
CMV, compared to the CB promoter. Previous studies have shown a 10-100 fold higher
promoter activity of CB promoter than that of CMV promoter depending on the vector dose.239,
266 This superior activity of CB promoter, noted in our study, even defeated the advantage of
dsAAV genome of the faster and stronger transgene expression. Importantly, the promoter
remained active after host cell differentiated into hepatocytes. Finally, site-specific integration
system of AAV vector may be employed. Considering the dilution of episomal rAAV vector
during cell division, site-specific integration AAV vector system may not only enhance but also
ensure a long-term transgene expression. Importantly, AAVS1 site has been reported to be a safe
integration site.46 A bipartite rAAV vector has been designed to fulfill this concept.101
In addition to the feature of easy isolation in large quantity from adipose tissue and
expendable in vitro, AT-MSCs, like bone-marrow derived MSCs, also exhibit
immunomodulatory and anti-proliferative effects on T cells.267, 268 Therefore, it is possible to
transplant allogeneic AT-MSCs from normal individual to AAT deficient patients without severe
immune response. This strategy can be tested using our recently-developed hAAT transgenic
mouse as donor. Transplantation of AT-MSCs from hAAT transgenic mice to genetically
mismatched recipients will not only avoid the ex vivo transduction, but provide better
understanding of hepatic differentiation of AT-MSCs.
In summary, results from our study using rAAV vector expressing hAAT gene consisted
and further extended the previous observations232 thus paved a path to both basic and clinical
studies.
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Figure 5-1. Experimental outline of AT-MSCs study. The recipients (female C57BL/6) were IP
injected twice (2 weeks interval) with 50mg/kg BW of monocrotaline (MCT) and received partial hepatectomy (PHx) to remove 70% of liver mass before transplantation. AT-MSCs were isolated from the femurs and tibias of male C57BL/6 mice. The newly purified BM cells were infected with rAAV1-CB- rAAV-CB-hAAT vector for 2 hours, washed three times with PBS, and transplanted into the recipient liver by intrasplenic injection. Serum hAAT levels were monitor by human AAT specific ELISA. Liver repopulation was measured by immunostaining.
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A.
B.
Figure 5-2. Characterization of AT-MSCs. (A) Expression of cell surface markers in AT-MSCs by immunofluorescence staining. (B) Multiple differentiation potential of AT-MSCs. Left, undifferentiated AT-MSCs; Middle, AT-MSCs were cultured for 2 weeks in an adipogenic induction medium and stained with Oil Red O for lipid droplets; Right, AT-MSCs were cultured for 2 weeks in an osteogenic induction medium and stained with Alizarin Red S (pH 4) for calcium phosphates.
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A
B
Figure 5-3. In vivo muscle or liver transduction by ssAAV and dsAAV vectors. C57BL/6 female
mice were injection 2×1010 particle rAAV vectors by intramuscular injection or portal vein injection to liver. A) In vivo muscle transduction by rAAV vectors. Solid square, ssAAV1-CB-hAAT vector; Open square, dsAAV1-CMV-hAAT vector; Solid triangle, saline group serve as negative control. B) In vivo liver transduction by rAAV vectors. Solid square, ssAAV8-CB-hAAT vector; Open square, dsAAV8-DHBV-hAAT vector; Solid triangle, saline group serve as negative control.
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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 (5×104cells/well, n=3) and infected 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, ssAAV-CB-hAAT vector; Cross, dsAAV-DHBV-hAAT vector; Dash, lower limit of quantification (LLOQ). hAAT level of PBS group ( negative control) was below LLOQ. (B) Double transduction of AT-MSCs by ssAAV1-CB-hAAT vector. Mouse AT-MSCs (passage=1) were seeded in 24-well (1×104cells/well, n=3) and infected with ssAAV1-CB-hAAT vector at 5x104 particles/cell. The accumulative hAAT in the culture medium was measured by hAAT ELISA. Triangle, one infection; Circle, two infections at 12 hr interval; Dash, lower limit of quantification (LLOQ). hAAT level of PBS group ( negative control) was below LLOQ.
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Figure 5-5. Detection of expression of human alpha 1-antitrypsin (hAAT) in recipient liver after transplantation of ssAAV1-CB-hAAT infected AT-MSCs by immunostaining. (A, C, D) Liver section from C57BL/6 mouse transplanted with ssAAV1-CB-hAAT infected AT-MSCs stained for hAAT (Brown). (B) Liver section from the same animal as in A, C, and D stained by anti-rabbit immunoglobulin G serving as negative control. (E) Liver section from normal human serves as positive control. (F) Liver section from an untransplanted C57BL/6 mouse serves as negative control.
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Figure 5-6. Detection of donor cells in recipient liver after AT-MSCs transplantation by fluorescence in situ hybridizations for Y-chromosome. (A) Male liver served as positive control. (B, C) Female mice treated with MCT/PHx and transplanted with AT-MSCs from male mouse. Y, Y chromosome.
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Figure 5-7. Detection of coexpression of human alpha 1-antitypsin (hAAT) and mouse albumin by immunostaining.(A) Liver section from mouse transplanted with ssAAV1-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 control. (D) Normal mouse liver section staining for mouse albumin served as positive control.
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Figure 5-8. Multi-organ homing of transplanted AT-MSCs. Tissue sections were from female C57BL/6 mouse at 8 weeks after transplantation with rAAV8-CB-GFP vector infected male AT-MSCs. (A) Spleen section stained for GFP (brown). (C) Lung section stained for GFP. (D) Bone section stained for GFP. Black arrowheads indicate the observed GFP staining. Images were viewed at ×20 magnification.
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Figure 5-9. Detection of expression of human alpha 1-antitrypsin (hAAT) in the serum. AT-MSCs from C57BL/6 mice were infected with ssAAV1-CB-hAAT vector at 5x104 particles/cells for 2 h and transplanted into liver of partially hepatectomized C57BL/6 recipient (1-2 x106 cells/mouse; n=3). The transgene expression was monitored by measuring the serum level of hAAT. Square is the serum from the treatment group. Dash is lower limit of quantification (LLOQ). The serum level of hAAT from untransplanted C57BL/6 mouse (negative control) was below the LLOQ.
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CHAPTER 6 SUMMARY AND FUTURE DIRECTION
Summary
Protein replacement therapy has been only optional treatment for AAT deficiency for more
than 20 years. Several new ideas have come up, implemented and tested for sake of the advances
in the field of molecular biology, disease pathogenesis, and biotechnology, etc. In the last 10
years, strides have been made in treating a secreted protein disorder such as AAT deficiency with
the use of rAAV gene therapy and bring it to clinical trials. At the meantime, stem cell has gone
from basic research to clinical application as regenerative medicine to increase our standard of
lives. For instance, bone marrow transplantation extends the lives of people suffering from
leukemia, lymphoma and other inherited blood disorders. A combination of gene therapy and
stem cell will broad their application than using either single therapy alone and will address
some intrinsic problem of gene therapy or stem cell therapy such as nonspecific targeting of viral
vector and differentiation potential of stem cells. In 1990, the first gene therapy trial was a cell-
based gene therapy. Patients’ T-cells were isolated and transduced with MoMLV-ADA vector ex
vivo, followed by returning to patients. Patients benefited and suffered no harmful effect from
this therapy, however, since T-cells have a limited life-span, patients need to receive periodic
infusion of their genetically –modified T cells. From this point, researches see the prospect of
using stem cell to develop a permanent cure, the stem cell-based gene therapy.
Our studies tested the feasibility of using adult stem cell-based gene therapy approach to
treat one of the common secreted protein disorders, AAT deficiency. We investigated two types
of viral vectors rAAV vector and lentiviral vector. rAAV vector is the safest viral vector among
all of the viral vectors, which leads to over 40 clinical trials involving 14 diseases and 4 serotype
rAAV vectors so far. Lentiviral vector is known by its high transduction efficiency and long-
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term transgene expression by integrating viral genome into host chromosome. Both viral vectors
represent unique property in the gene therapy. To answer the question that whether genetically
modified adult stem cells can be utilized to correct the genetic defect, we first transduced liver
progenitor cells (oval cells) with rAAV and lentiviral vector expressing hAAT and transplanted
these cells into the mouse liver. Results from these studies showed that oval cells can be
transduced by both rAAV and lentiviral vectors. Transgene (hAAT) expression can be detected
in the recipient liver and transgene produce could be secreted into the circulation to boost serum
hAAT level.
Results from oval cell study were promising and indicated it is feasible to use adult stem
cell for liver gene delivery. However, isolation of oval cells is not clinically applicable, although
oval cells can be isolated from animal in large quantity. In order to avoid this problem, in the
second set of studies, we transduced and transplanted BM cells into mouse livers. BM cells have
been proved to be capable of converting into hepatocytes and contributing to liver regeneration.
More importantly, BM cells can be used for autologous transplantation to eliminate rejection
problem from allograft transplantation. These results showed that BM cells can be transduced by
rAAV and lentiviral vectors and engrafted into liver resulting in transgene expression and
transgene product in the serum. rAAV8 vector demonstrated superior transduction efficiency
than rAAV1 vector and lentiviral vector.
To obtain enough cell number from BM for clinical application is challenging. Adipose
tissue represents an ideal source of autologous stem cells, AT-MSCs. Liposuction results in
minimal patient discomfort and adipose tissue can be obtained in large volume for yielding
enough cells for clinical practice. Hence, we performed the third set of studies using AT-MSCs.
Results demonstrated that AT-MSCs can be efficiently transduced by rAAV 1 vector. Ex vivo
107
transduced and transplanted AT-MSCs can mediate transgene expression in the liver and result
in sustained transgene product, hAAT, in the circulation. Importantly, engrafted AT-MSCs
presented hepatocyte cell function e.g. albumin production.
In conclusion, this study showed adult stem cells can serve as carrier for gene delivery.
Adult stem cell engrafted into target organ and played as a platform for transgene expression
after transplantation. Adult stem cell-based gene therapy presents a novel approach for treatment
of human genetic disease. In this study, we have develop two stem cell (BM and AT-MSC)
based gene therapies for the treatment of AAT deficiency.
Future Direction
Achieving therapeutical serum level of transgene product (hAAT) is the final goal.
Efficiency of adult stem cell-based gene therapy is determined by three main factors,
transduction efficiency, engraftment efficiency, and transgene expression. Optimization in any of
these three factors will definitely contribute to achieve our goal. For increasing transduction
efficiency of viral vectors, engineering viral capsid and genome have been carried out. Tissue
and cell specific targeting and site-specific integration are the two attractive properties that future
viral vectors want to pursuit. Specific targeting to a special cell type will decrease the possibility
of site effect resulting from nonspecific binding to unwanted cells. At the mean time, it will
enhance the viral vectors concentration in the targeted cells and leads to increase transgene
expression. Site-specific integration provides a long-term treatment and reduces the risk of
tumorigenesis resulting from randomly integrating into promoter region of oncogene.
Clarifying and understanding of stem cell homing will in turn aid application of stem cell
transplantation. But stem cell homing and engraftment are a complex and multistep process.
These evolve various adhesion receptors and ligands that mediate cell-to-matrix and cell-to-cell
interaction, including selectins, integrins, and Ig family and numbers of others undefined factors.
108
Several signaling pathways (e.g.SDF1-α/CXCR4 signaling) have been put forward, but more
researches are needed to solve this puzzle. Tumor formation is a big issue for stem cell therapy.
Detail examination of the tumorigenesis of stem cell is required to make stem cell an effective
and safe therapy.
109
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BIOGRAPHICAL SKETCH
Hong Li was born in Fuzhou, Fujian Province, P. R. China in 1979. After graduation from
high school at Fuzhou No. 8 Middle School in 1997, she started her college education at China
Pharmaceutical University in Nanjing, P. R. China. She was awarded her bachelor’s degree in
biotechnical pharmaceutics in 2001 and master’s degree in microbiology and biochemical
pharmacy in 2004. She then joined the Department of Pharmaceutics at University of Florida and
began her doctoral study under the guidance of Dr. Sihong Song in August 2004. Her research
focused on adult stem cell-based gene therapy for alpha 1-antitrypsin deficiency. At the
meantime, she obtained her master’s degree in statistics in 2008 from University of Florida,
Department of Statistics.