baculovirus-mediated gene delivery for ...glioma cells in the context of baculovirus. the transgene...

BACULOVIRUS-MEDIATED GENE DELIVERY FOR

GLIOMA THERAPY

LI FENG

NATIONAL UNIVERSITY OF SINGAPORE

2006

BACULOVIRUS-MEDIATED GENE DELIVERY FOR

GLIOMA THERAPY

LI FENG

(B. Sc.)

A THESIS SUBMITTED FOR THE DEGREE OF

MASTER OF SCIENCE

DEPARTMENT OF BIOLOGICAL SCIENCES

NATIONAL UNIVERSITY OF SINGAPORE

AND

INSTITUTE OF BIOENGINEERING AND NANOTECHNOLOGY

2006

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ACKNOWLEDGMENTS

I would like to take this opportunity to extend my deepest gratitude to my

supervisor Dr. Wang Shu, Group Leader, Institute of Bioengineering and

Nanotechnology; Associate Professor, Department of Biological Science,

National University of Singapore, for his continuous support, patient guidance

and stimulating discussion.

I am also grateful to my colleagues in the Institute of Bioengineering and

Nanotechnology for their assistance and companionship throughout my study

in Singapore.

Special acknowledgments go to Dr. Wang Chaoyang and Dr. Ong Seow

Theng for their help with the research project.

Special thanks to Dr. Zeng Jieming and Dr. Jurvansuu Jaana for their

critical review of the manuscript.

This thesis is dedicated with affection to my parents in China, whose

courage and patience have always been an inspiration to me.

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TABLE OF CONTENTS

ACKNOWLEDGMENTS .................................................................................. I

TABLE OF CONTENTS .................................................................................. II

SUMMARY .....................................................................................................IV

LIST OF FIGURES..........................................................................................V

ABBREVIATION ...........................................................................................VII

Chapter One: Introduction ............................................................................ 1

1.1 Gliomas: the terminator ............................................................................. 2

1.2 Glioma gene therapy: a novel strategy ...................................................... 3

1.3 Baculovirus: an emerging vector for gene therapy .................................... 6

1.4 Control the gene expression at the transcriptional level ............................ 9

1.4.1 Glioma-specific promoter .................................................................... 9

1.4.2 Expression cassette for siRNA.......................................................... 13

1.5 Glioma animal model and non-invasive imaging ..................................... 15

1.6 Objectives of the study ............................................................................ 20

Chapter Two: Materials and Methods ........................................................ 21

2.1 Cell lines and experimental animals ........................................................ 22

2.2 Shuttle plasmids and recombinant baculovirus production...................... 23

2.3 Virus transduction.................................................................................... 27

2.4 Luciferase activity assay.......................................................................... 28

2.5 Detection of eGFP expression................................................................. 30

2.6 RT-PCR ................................................................................................... 30

2.7 Fluorescence immunohistochemistry ...................................................... 32

2.8 Cell viability assay ................................................................................... 33

2.9 Rat C6 glioma xenograft model and tumor growth monitoring................. 34

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Chapter Three: Results ............................................................................... 36

3.1 Establishment of C6 glioma xenograft model .......................................... 37

3.2 DTA expressing baculovirus-mediated inhibition of glioma cell growth.... 40

3.2.1 Effective transduction of glioma cells by baculoviral vectors............. 40

3.2.2 Modified GFAP promoters improve transgene expression to glioma

cells................................................................................................... 43

3.2.3 Inhibition of protein synthesis and glioma cell growth in vitro............ 49

3.2.4 Expression of reporter genes in glioma xenograft ............................. 55

3.2.5 Inhibition of glioma xenograft growth................................................. 58

3.3 siRNA expressing baculovirus-mediated gene silencing ......................... 61

3.3.1 Knockdown of luciferase gene expression in cultured cells .............. 61

3.3.2 Knockdown of luciferase gene expression in rat brain ...................... 66

Chapter Four: Discussion and Conclusion ............................................... 68

References ................................................................................................... 77

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SUMMARY

Gene therapy is a promising therapeutic strategy for gliomas, which are

incurable by conventional approaches. The success of gene therapy is greatly

dependent on delivery vectors. In the current study, we investigated the

feasibility of using insect baculovirus as a gene delivery vector for glioma

therapy. A glial-specific promoter was created by addition of a cytomegalovirus

(CMV) enhancer upstream to a glial fibrillary acidic protein (GFAP) promoter.

This expression cassette showed a high level expression of reporter genes in

glioma cells in the context of baculovirus. The transgene expression level was

further improved by flanking the expression cassette with inverted terminal

repeats from adeno-associated virus. When therapeutic gene encoding

diphtheria toxin A-chain was used, the inhibition of glioma cell growth was

demonstrated in cell lines and in a rat C6 glioma xenograft model. RNA

interference mediated by a recombinant baculoviral vector with a hybrid

promoter (CMV enhancer/H1 promoter) was also studied and an effective

knockdown of target gene expression was observed. These results show that

baculoviral vectors might provide a new effective option for cancer gene

therapy.

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LIST OF FIGURES

Fig. 1 Monitoring the C6 glioma xenograft model by calculating the tumor size. Fig. 2 Monitoring the C6 glioma xenograft model by luciferase activity assay. Fig. 3 Transduction of glioma cells with baculovirus with luciferase reporter

gene. Fig. 4 Transduction of glioma cells with baculovirus with eGFP reporter gene. Fig. 5 Modified GFAP promoters improved baculovirus-mediated luciferase

expression in glioma cells. Fig. 6 Modified GFAP promoters improved baculovirus-mediated eGFP

expression in glioma cells. Fig. 7 RT-PCR analysis of DTA expression. Fig. 8 BV-CG/ITR-DTA mediated inhibition of protein synthesis in cultured

glioma cell lines. Fig. 9 BV-CG/ITR-DTA mediated inhibition of protein synthesis in C6-Luc cell

line. Fig. 10 BV-CG/ITR-DTA mediated selective inhibition of glioma cells growth in

vitro. Fig. 11 In vivo eGFP reporter gene expression in gliomas mediated by

baculovirus carrying the hybrid CMV E/GFAP promoter and ITRs. Fig. 12 In vivo luciferase gene expression in gliomas mediated by baculovirus

carrying the hybrid CMV E/GFAP promoter and ITRs. Fig. 13 Monitoring the C6 glioma xenograft growth in the rat brain by luciferase

activity assay. Fig. 14 Monitoring the C6 glioma xenograft growth in the rat brain by BLI. Fig. 15 Baculovirus-mediated gene silencing effects in vitro. Fig. 16 Quantitative analyses of baculovirus-mediated gene silencing effects in C6 cells.

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Fig. 17 Quantitative analyses of baculovirus-mediated gene silencing effects in

NT2 cells. Fig. 18 Baculovirus-mediated silencing effects in rat brain.

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ABBREVIATION

AAV adeno-associated virus

BBB blood brain barrier

BLI bioluminescence imaging

BV Baculovirus

CAG CMV enhancer/β-actin promoter

CMV Cytomegalovirus

CMV E enhancer of cytomegalovirus immediate-early gene

CNS central nervous system

DMEM Dulbecco’s modified eagle’s medium

DTA diphtheria toxin A-chain

EF1α elongation factor 1 α

eGFP enhanced green fluorescence protein

EGFR epidermal growth factor receptor

GBM glioblastoma multiforme

GCV Ganciclovir

GFAP glial fibrillary acidic protein

HSV herpes simplex virus

HSV-tk herpes simplex virus thymidine kinase

IACUC institutional animal care and use committee

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ITR inverted terminal repeats

LTR long terminal repeats

Luc Luciferase

MBP myelin basic protein

MCS multiple cloning site

MOI multiplicity of infection

MRI magnetic resonance imaging

NIRF near-infrared fluorescence

PBS phosphate-buffered saline

PDGF human platelet-derived growth factor

PET positron emission tomography

PFU plaque-forming units

PSE proximal sequence element

RISC RNA-induced silencing complex

RLU relative light unit

RNAi RNA interference

shRNA short hairpin RNA

siRNA small interfering RNA

snRNA small nuclear RNA

TH tyrosine hydroxylase

VEGF vascular endothelial growth factor

Chapter One: Introduction

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Chapter One

Introduction


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1.1 Gliomas: the terminator

Gliomas are a collection of tumors that mainly originate from transformed

glial cells, the supporting cells in the central nervous system (CNS; Holland,

2000). Although the incidence is about 3 per 100000 people per year

(DeAngelis, 2001), gliomas remain among the most devastating forms of

human cancers. According to their malignancy, gliomas are clinically divided

into four grades, among which grade 4 glioblastoma multiforme (GBM)

accounts for half of all brain tumors and is the most invasive and aggressive

form. Conventional therapeutic approaches such as surgery, chemotherapy,

and radiotherapy, though progressing well in the past few decades, are still not

able to effectively cure GBM, and most patients die 12-18 months (Surawicz et

al., 1998) after diagnosis. The reason for the failure of treatment is inherent to

the properties of gliomas, which are “multiforme” grossly, microscopically and

genetically. In addition, gliomas are highly proliferative, highly vascularized,

and aggressively infiltrative into the brain (Holland, 2000). Gliomas have also

evolved a mechanism to escape from immune surveillance (Sikorski et al.,

2005). The outcome of surgery is often unsatisfactory, because it is difficult to

completely dissect the tumors and the surgical operations in the brain often

result in neurological complication. For the radiotherapy, the radiation dose

required to kill gliomas is much higher than can be tolerated by normal brain

tissues, and increased radiation dose is always associated with the occurrence

of undesirable tissue damage. The failure of chemotherapy results partially


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from the blood brain barrier (BBB), which hinders the transport of many

chemical drugs, and thus makes it difficult to achieve an effective drug

concentration in the brain to kill the glioma cells. Moreover, the appearance of

chemo-resistant glioma cells makes it more difficult to treat.

1.2 Glioma gene therapy: a novel strategy

Because of the poor outcome of conventional approaches, great

expectation has been set on novel therapeutic strategies such as gene therapy

for the treatment of gliomas. Initially discussed during the 1960s and the 1970s

(Friedmann, 1992), gene therapy is defined as the correction of missing genes,

replacement of defective genes, removal or down regulation of abnormal

genes. The inherited single gene disorder was the initial target of gene therapy,

and evidence has accumulated that it can be used for the treatment of various

diseases including hemophilia (Walsh, 2003), lysosomal storage disorders

(Cheng et al., 2003), severe combined immunodeficiency (Gaspar et al., 2003),

diabetes mellitus (Yechoor et al., 2005), cancer (McNeish et al., 2004), etc.

Since the first gene therapy clinical trial for patients with gliomas was carried

out more than a decade ago (Oldfield et al., 1993), many therapeutic

modalities for gliomas have been proposed and investigated (Barzon et al.,

2006; Pulkkanen et al., 2005), among which are suicide gene therapy, genetic

immunotherapy, tumor suppressor gene or oncogene approaches, and

anti-angiogenesis gene therapy.


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Suicide gene therapy is one of the commonly employed therapeutic

approaches, accounting for 73% of the approved glioma gene therapy clinical

trials (Barzon et al., 2006). As an attractive candidate for suicide gene therapy,

the diphtheria toxin A-chain (DTA) gene has been extensively studied by

several groups (Ayesh et al., 2003). Secreted by Corynebacterium diphtheriae

as a precursor polypeptide, diphtheria toxin is composed of two fragments, the

A and B chains. The B chain contains a binding domain which interacts with

the receptors present on the surface of most eukaryotic cells and facilitates the

cell uptake of the A chain into cytoplasm (Collier, 1975). Once inside the

cytoplasm, the A chain will catalyze the ADP-ribosylation of diphthamide

residue present in the eukaryotic elongation factor 2, which lead to inhibition of

host cell protein synthesis and eventually result in the cell death (Choo et al.,

1994; Sandvig et al., 1992). Only a low concentration of DTA is required to

cause cell death through a cell cycle-independent pathway (Yamaizumi et al.,

1978; Rodriguez et al., 1998).Thus, the DTA gene is superior to other

candidate genes such as herpes simplex virus thymidine kinase (HSV-tk) gene,

which requires administration of prodrugs and whose efficacy is often

undermined by the low prodrug concentration achieved within the glioma cells

in the brain. In addition, the DTA gene, encoding DTA, but not DTB, has

already been cloned and engineered for expression in mammalian cells.

Without the B chain, DTA released after cell death is unable to enter the nearby

cells, thus preventing unwanted toxicity to normal tissues.


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The use of RNA interference (RNAi) technique for glioma gene therapy is

another recently developed strategy. RNAi was first described in C. elegans as

a response to exogenous double-stranded RNA (Fire et al., 1998) and has

subsequently been demonstrated in diverse eukaryotes such as insects, plants,

fungi, and vertebrates. As a highly specific posttranscriptional gene silencing,

RNAi is a powerful tool for functional genomic study, generating animal models,

as well as in the treatment of many diseases such as viral infections and

cancer. (Novina et al., 2004; Pardridge, 2004; Spankuch et al., 2005). The use

of RNAi-based approaches for glioma therapy has been summarized in a

recent review (Mathupala et al., 2006). Since the activation or over-expression

of various genes related to cell-adhesion/motility and invasiveness, growth

factors and/or their receptors is usually associated with the development of

gliomas (Mathupala et al., 2006; Barker et al., 1995), knockdown the

expression of these molecules such as vascular endothelial growth factor

(VEGF; Tao et al., 2005), telomerase (Pallini et al., 2006), or epidermal growth

factor receptor (EGFR; Kang et al., 2006; Saydam et al., 2005), could be an

effective treatment of gliomas.


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1.3 Baculovirus: an emerging vector for gene therapy

It is impossible to obtain success in gene therapy without effective gene

delivery systems that can achieve high levels of therapeutic gene expression

in targeted cells. Gene delivery vectors can be classified into viral and

non-viral vectors. Non-viral gene delivery systems include: cationic polymer

complexes (De Smedt et al., 2000), liposomes (Simoes et al., 2005), micelles

(Adams et al., 2003) and nanoparticles (Panyam et al., 2003). Tremendous

efforts have been made in the study of non-viral vectors, for several reasons.

First, compared with viral vectors, non-viral vectors are less likely to induce an

immune response and thus can be administered repeatedly to the patient

without causing severe adverse effects or being neutralized by preexisting

antibodies. Secondly, they are relatively easily manufactured as

pharmaceutical products. However, the low transfection efficiency of non-viral

vectors remains a notorious obstacle that needs to be overcome before use in

clinical application. In contrast, high transduction efficiency is a distinct

property of viral vectors such as retrovirus (Weber et al., 2001), adenovirus

(McConnell et al., 2004), adeno-associated virus (Conlon et al., 2004), herpes

simplex virus type 1 (Epstein et al., 2005), or lentivirus (Copreni et al., 2004).

Viruses have evolved smart mechanisms to enter host cells and utilize the host

cells’ machinery to survive. Owing to these mechanisms, which confer the viral

vectors’ incomparably high transduction efficiency, viral vectors remain

predominant in gene therapy clinical trials. However, the application of viral


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vectors is also hindered by several shortcomings, including limited

DNA-carrying capacity, insertional mutagenesis and immunogenicity. The

death of a teenage from an immune reaction to the adenovirus vector during

the clinical trial carried out at the University of Pennsylvania presents an

example of the problems with viral vectors, one which even caused a setback

in the gene therapy researches (Check, 2005).

Recently, the baculovirus (Autographa californica multiple

nucleopolyhedrovirus) based vectors, traditionally used as biopesticides

(Tomalski et al., 1991) have emerged as novel gene delivery vectors with

many attractive features (Ghosh et al., 2002; Kost et al., 2005). Firstly,

baculovirus has an excellent biosafety profile. As an insect virus, it will not

replicate or recombine with preexisting genetic materials in mammalian cells

and shows no obvious pathogenicity in targeted cells (Ghosh et al., 2002).

Secondly, baculovirus is able to accommodate as much as 100 kb or more

DNA insert and its whole genomic sequence has been determined, providing

many conveniences for genetic manipulation. The large cloning capacity

enables the delivery of a large functional gene or several genes within a single

vector. Thirdly, several commercially available techniques for preparing

baculovirus have been developed and large amounts of high titer baculovirus

can be easily prepared in serum-free culture media. This feature paved the

way for scaling up its manufacture in the pharmaceutical industries and the

use of serum-free media avoided the potential danger of contamination from


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the serum of donating animals. Last but not least, compared with other viral

vectors such as adenovirus, the lack of preexisting immune response against

baculovirus provides an additional advantage for the use of baculovirus in vivo.

The recombinant baculoviruses with mammalian expression cassettes were

able to deliver transgenes into a broad range of cells including primary rat

chondrocytes (Ho et al., 2004), mouse primary kidney cells (Liang et al., 2004),

hepatic stellate cells (Gao et al., 2002), human osteosarcoma cell lines (Song

et al., 2001), human mesenchymal stem cells (Ho et al., 2005) and human

embryonic stem cells (manuscript in preparation). The in vivo transgene

expression profile of recombinant baculoviruses could be controlled by the

route of administration and expression cassettes (Li et al., 2005; Li et al., 2004).

The use of recombinant baculovirus for human prostate cancer gene therapy

has been described (Stanbridge et al., 2003). Another recent study has

explored the use of recombinant baculovirus for RNAi (Nicholson et al., 2005),

which indicated that a recombinant baculovirus containing the U6 promoter

was able to knock down targeting mRNA and protein effectively, suggesting

baculovirus might be an alternative short hairpin RNA (shRNA) delivery system

without the problems associated with other viral vectors. However, despite a

good understanding of all these attractive features of baculovirus, most of the

studies of baculovirus still remain at the stage of reporter gene delivery, and its

application to glioma gene therapy has not been reported, even in a preclinical

study.


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1.4 Control the gene expression at the transcriptional level

An expression cassette, mainly composed of promoters and other

regulatory elements, is an important factor that controls the magnitude,

duration, and specificity of gene expression at the transcriptional level. The

promoter is the main regulator of gene expression, and can be classified into

three categories: viral promoter, cellular promoter and hybrid promoter. Other

regulatory elements include the posttranscriptional regulatory element of

woodchuck hepatitis virus（Hlavaty et al., 2005）, inverted terminal repeats (ITR)

of AAV（Chikhlikar et al., 2004; Xin et al., 2003）, and the central polypurine

tract (Van Maele et al., 2003). The manipulation of the gene expression

cassette enables us to achieve optimal expression profiles for particular

therapeutic applications.

1.4.1 Glioma-specific promoter

Due to their high transcriptional activity, viral promoters, such as

cytomegalovirus (CMV) major immediate-early promoter/enhance, have been

used to achieve robust transgene expression (Kaplitt et al., 1994; McCown et

al., 1996). However, the application of viral promoters for glioma therapy was

restricted by their non-specific gene expression properties. For example, after

injection into the rat striatum of an AAV vector, where the tyrosine hydroxylase

(TH) gene is under the control of a CMV promoter, the expression of the TH

gene in neurons was observed (Kaplitt et al., 1994). The untargeted gene


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expression in neurons, though desirable for the treatment of many neuron

degenerative diseases such as Parkinson’s disease and Alzheimer's disease,

will become a serious issue, particularly when toxin genes for glioma therapy

are used, since the expression of toxin genes in neurons, which have

important physiological functions, will cause severe adverse effects in the CNS.

Therefore, the universal viral promoters have gradually been replaced by other

recently developed glioma or tumor specific promoters in the glioma gene

therapy.

Unlike the viral promoters, the cellular promoters have specificity in driving

the transgene expression, making it possible to target the transgene

expression within glioma cells and hence avoid adverse effects caused by the

over-expression of therapeutic genes in non-targeted normal tissues.

Candidate promoters for glioma therapy could be tissue-specific promoters

such as the glial fibrillary acidic protein (GFAP; Vandier et al., 2000; Vandier et

al.,1998; Ho et al.,2004; Zamorano et al.,2004) and myelin basic protein

promoters (Shinoura et al., 2000; Miyao et al., 1993; Miyao et al., 1997);

promoters targeting tumor endothelium (Pore et al., 2003) and tumor-specific

promoters, such as the nestin promoter (Lamfers et al., 2002), survivin

promoter (Kleinschmidt-DeMasters et al., 2003), and E2F-1 promoter (Parr et

al.,1997), which are highly active in many cancer cells as well as in glioma

cells. The GFAP promoter is a promising candidate for glioma-targeted gene

expression. It is active in glial cells and gliomas as well, but not, in neurons. A


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recombinant adenovirus carrying HSV-tk gene under the control of the GFAP

promoter demonstrated higher level of HSV-tk expression in rat C6 glioma cell

line than in the non-glial MDA-MB-231 cell line. The subsequent treatment with

the HSV-tk prodrug ganciclovir (GCV) showed high toxicity in two glial cell lines

(C6, U251), but low toxicity in the non-glial cell lines tested (Vandier et al.,

2000). This strategy has also been tested in a retroviral vector in which the

expression of a full-length human growth arrest specific 1(gas1) cDNA is under

the transcriptional control of a human GFAP promoter (gfa2). It was observed

that the expression of gas1 caused cell death in vitro and inhibits tumor growth

in vivo in a transplanted tumor model, by triggering apoptosis (Zamorano et al.,

2004).

Despite the good cell type specificity of the GFAP promoter, its application

is curbed by its low transcriptional activity, which, in most cases, is not

sufficient for glioma therapy. Therefore, further improvements are required to

enhance the transgenes expression (de Leeuw et al., 2006). An enhanced

GFAP promoter was created by inserting three additional copies of putative

GFAP enhancer regions. Compared with original GFAP promoter, this hybrid

promoter gave 75-fold higher LacZ expression on plasmid transfection into

U251 cells and approximately 10-fold higher LacZ expression in the context of

an adenoviral vector (de Leeuw et al., 2006). In addition, when the adenoviral

vector containing this enhanced promoter was injected into the brain of nude

mice (de Leeuw et al., 2006), targeted LacZ expression in GFAP positive cells


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was observed.

The hybrid promoter composed of a cellular promoter appended with a

viral enhancer/promoter has been proved to be a successful approach to

improve transcriptional activity. The CMV enhancer/beta-actin (CAG) promoter

is a good example. Widely employed in gene therapy, the CAG promoter is a

robust constitutive promoter composed of the CMV enhancer fused upstream

to the chicken beta actin promoter (Sawicki et al., 1998; Xu et al., 2001).

Administered through portal vein injection, an AAV vector with the CAG

promoter showed 137-fold higher human factor X expression in mouse livers

than those with the CMV promoter/enhancer (Xu et al., 2001). Besides the

improvement of transcriptional activity, the retention of cell type specificity is

also a critical issue. The specificity of this type of hybrid promoter combination

has been evaluated in previous study. A hybrid promoter-CMV E/PDGF

promoter-has been constructed by appending the human platelet-derived

growth factor (PDGF) promoter downstream to a 380-bp fragment of the CMV

enhancer. When it was employed in the context of plasmid, AAV-2 vectors, or

baculoviral vectors (Liu et al., 2004; Wang et al., 2005; Li Y et al., 2005),

improved transgene expression in neuronal cell lines has been achieved

compared with the vector containing the original PDGF promoter, while a low

expression level was observed in non-neuronal cell lines. After injection into

the rat brain, this hybrid promoter demonstrated a neuronal specificity, driving

luciferase reporter gene expression almost exclusively in neurons.


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The hybrid promoter might also be a useful approach to improve the

relatively low transcriptional activity of the GFAP promoter, while retaining the

cell type specificity, thus creating a suitable promoter for glioma gene therapy.

1.4.2 Expression cassette for siRNA

Chemically synthesized small interfering RNA (siRNA) duplexes of 21-23nt

can be delivered into the cytoplasm where they are recruited into the

RNA-induced silencing complex (RISC) and then trigger the cleavage of target

mRNA in a sequence-specific manner. Because of the poor intracellular

stability of siRNA, a more effective way is to use vector-based siRNA

expression systems that can constitutively express the shRNA (Wadhwa et al.,

2004). shRNA is processed by Dicer, an RNase III-related ribonuclease, into

siRNA, which then results in silencing of a target gene (Stanislawska et al.,

2005; Fire et al., 1998). Three types of promoters, including Pol III promoter,

Pol II promoter, or inducible Pol III promoter can be used in siRNA expression

(Arendt et al., 2003). The Pol III promoter is in charge of the transcription of

genes encoding tRNAs, 5S rRNA, and an array of small, stable RNAs (Harvey

et al., 2003). The rationale for using the Pol III promoter in the siRNA

expression cassettes is that Pol III transcripts are abundant in human cells

(Thompson et al., 1995). Pol III promoters can be further classified into three

categories (type I, type II and type III; Paule et al., 2002), and the two popular

Pol III promoters, the human U6 small nuclear RNA (snRNA) promoter and the


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human H1 promoter are both type III promoters. The transcriptional activities of

the three types of Pol III promoters vary with the composition of promoter

elements, the promoter position relative to the transcriptional start site, the

location of a promoter within a given vector, and probably also the type of cells

tested (Arendt et al., 2003; Ilves et al., 1996; Boden et al., 2003; Koper-Emde

et al., 2004). Therefore, the choice of a Pol III promoter is a crucial issue in the

design of shRNA expression cassettes for vector-based RNAi. Alternatively,

the transcriptional activity of Pol III promoter can be improved through

modification (Thompson et al., 1995; Paul et al., 2002). For example, the CMV

enhancer has been employed in one study to improve the activity of U6

promoter. When the CMV enhancer was placed near the U6 promoter in the

context of a plasmid, increased shRNA expression and enhanced silencing of

the target gene was observed (Xia et al., 2003). However, an apparent

decrease in U6 RNA half-life was accompanied with an increased dose of U6

gene construct (Noonberg et al., 1996), suggesting the existence of an

intracellular regulatory mechanism to prevent over-accumulation of U6 RNAs.

This finding raises concerns regarding the use of U6 promoters for high-level

expression of shRNA. In the current study, we focused on the H1 promoter, a

Pol III promoter that is responsible for the transcription of a unique gene

encoding the RNA component of the nuclear RNase P that cleaves tRNA

precursors into mature 5’-termini (Myslinski et al., 2001). The H1 promoter has

four cis-acting elements that are essential for maximal expression, located


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within 100 bp of the 5’-flanking region. They are characterized by an unusually

compact structure with the octamer motif and staf binding site near the

proximal sequence element (PSE) and TATA motif (Myslinski et al., 2001). A

hybrid promoter was constructed by fusing a 380bp fragment of the CMV

enhance upstream to the H1 promoter, then in vitro and in vivo experiments

were carried out to test if this modified H1 promoter was able to enhance the

gene silencing effects.

1.5 Glioma animal model and non-invasive imaging

A particular glioma gene therapy protocol cannot be tested in human

clinical trials until it has been verified on preclinical small laboratory animal

glioma models. The routinely used tumor models are created by implanting

glioma cells either into the brains of experimental animals (orthotopic model) or

into the flank subcutaneously (heterotopic model). A good glioma model should

have a well-defined in vivo growth profile which resembles the growth of

human gliomas in the brain. In addition, its response to treatment should be

similar to that of the human gliomas. Although, till now, there has not been a

perfect animal glioma model which could exactly mimic the real human

gliomas, currently available models have provided useful tools for the

evaluation of glioma gene therapy approaches, among which the C6/Wistar rat

intracerebral glioma model is one routinely used model for many studies (Barth,

1998; Zhang et al., 2002).


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For the success of glioma gene therapy studies, it is also crucial to develop

techniques to monitor the growth of gliomas in vivo. There are many

conventional methods including measuring the tumor size with caliper or

weighing the tumor after dissection. Although these traditional methods are

straightforward and reliable in some circumstances, their applications were

restricted by several reasons. There is a large variation in the measurement of

tumor size with caliper and it is also impossible to directly measure the tumor

growth of orthotopic glioma xenograft growing in the brain. When the tumor

weight is used as a parameter, animals have to be sacrificed before each

measurement. This endpoint measurement usually increases the amount of

experimental animals needed for statistical analysis. Therefore, many

researchers are devoted to the development of novel molecular imaging

approaches, such as magnetic resonance imaging (MRI; Immonen et al.,2004;

Hamstra et al., 2004), positron emission tomography (PET; Yaghoubi et al.,

2005), and near-infrared fluorescence (NIRF) imaging (Ntziachristos et al.,

2002; Weissleder et al.,1999; Becker et al.,2001; Ntziachristos et al., 2004).

MRI is a technique that is already used in clinics. The high spatial resolution,

excellent soft tissues differentiation, and the ability to measure multiple

physiological and metabolic parameters make it an important tool in the

diagnosis and treatment of patients with gliomas. To facilitate imaging, a

contrast agent is usually injected before MRI scanning. Recently developed

physiologic and metabolic MRI (Cao et al., 2006), magnetic resonance


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spectroscopy (Nelson, 2003), perfusion-weighted MRI, and proton

spectroscopic MRI (Law et al., 2005) can provide more sophisticated

information and will further benefit the treatment of gliomas. PET is another

imaging approach based on the detection of positron-emitting molecular

probes labeled with isotopes such as 18F, 11C, 15O, and 124I. For example, PET

scanning of 18F-fluorodeoxyglucose was used to assess tumor cell viability and

therapeutic efficacy of HSV-tk suicide gene therapy in C6 glioma model

(Yaghoubi et al., 2005). NIRF imaging is able to image deeper tissues such as

intracranial gliomas and get 3-D information (Weissleder et al., 1999) and its

good performance is attributable to the low background autofluorescence and

the high tissue-penetrating ability of the near-infrared spectrum (700-900nm)

used in imaging. Despite the progress of these techniques, none has yet been

established as a “gold standard” method, especially in pre-clinical animal

studies.

In the past few years, the introduction of bioluminescence imaging (BLI) as

a complementary experimental imaging technique for small animals has

achieved satisfactory progress. Bioluminescence is the visible light

(400-620nm) emitted during the oxidation of particular substrate that is

catalyzed by luciferase. Current available luciferase reporter genes include:

bacterial Lux genes of terrestrial Photorhabdus luminescens and marine Vibrio

harveyi bacteria; eukaryotic luciferase Luc gene from firefly species (Photinus);

eukaryotic luciferase Ruc gene from the sea pansy (Renilla reniformis). The


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firefly luciferase gene is most widely used to quantify gene expression (Soling

et al., 2004; Rehemtulla et al., 2002; Ray et al., 2004; Iyer et al., 2004). For

animal imaging, an instrument composed of a light-tight chamber and a highly

sensitive CCD camera is used to detect the bioluminescence, mainly the red

component of emission spectrum, penetrating the tissues and provides

quantitative information. When the tumor cells are genetically engineered to

stably express luciferase genes, the progress of the tumor and its response to

treatment can be non-invasively and quantitatively monitored in vivo (Caceres

et al., 2003; Jenkins et al., 2003; Uhrbom et al., 2004). When bioluminescent

PC-3M-Luc-C6 human prostate cancer cells were transplanted

subcutaneously in a mouse tumor model, a good correlation between

bioluminescence signal and tumor size measured by caliper was observed

(Jenkins et al., 2003). The bioluminescence signal also correlated well with the

total lung weight in an A549-Luc lung colonization model (Jenkins et al., 2003).

Owing to its high sensitivity, the detection of tumor metastasis has also been

demonstrated in an HT29 spontaneous metastatic tumor model (Jenkins et al.,

2003). The application of BLI in the intracranial glioma models is particularly

attractive. By using a luciferase-expression 9L glioma cell, 9L-Luc intracranial

glioma models have been established, allowing non-invasive monitoring of the

tumor response to chemotherapy (Rehemtulla et al., 2000) and photodynamic

therapy (Moriyama et al., 2004). Excellent correlation (r = 0.91) between

photons detected by BLI and tumor volume measured by MRI was


- 19 -

demonstrated (Rehemtulla et al., 2000). The application of BLI and luciferase

stable expression glioma models have several advantages: generating

luciferase-stable cell lines is technically simple; the tumor growth before and

after treatment can be monitored continuously in a real-time manner in

individual animals thus reducing the subject-to-subject variation and

minimizing the number of animals needed in the test; and the commercial

available imaging system for BLI is more affordable than the expensive

instruments for MRI and PET. Therefore, BLI has been increasingly applied in

preclinical animal studies.


- 20 -

1.6 Objectives of the study

The purpose of this study was to investigate the possibility of using a novel

recombinant baculoviral vector for glioma gene therapy. The expression

cassette is one crucial element in the vector that determines the magnitude,

duration, and location of gene expression on transcriptional level. Thus the

expression cassette could be manipulated to improve the expression profile of

the gene delivery system. To target the expression of a toxin gene into glioma

cells, we constructed a recombinant baculovirus with a GFAP promoter-based

expression cassette. The expression profile of this baculoviral vector carrying

reporter genes have been characterized in both in vitro and in vivo studies. In

addition, the therapeutic effects have been evaluated in glioma cell lines and in

a C6/Wistar glioma model. We also explored in the current study whether a

recombinant baculovirus harboring a hybrid CMV E/H1promoter could be used

for RNAi and evaluated the silencing effects in cultured cells and in

experimental animals. This study on baculovirus will benefit the development

of gene delivery vectors for glioma gene therapy and provide useful preclinical

information required for future clinical trials.

Chapter Two: Materials and Methods

- 21 -

Chapter Two

Materials and Methods


- 22 -

2.1 Cell lines and experimental animals

Human glioma cell lines (BT325, U251, U87, H4, SW1783, and SW1088),

rat glioma cell lines C6, two non-glioma cell lines (HepG2 and NIH3T3), and

NT2 human neural precursor cell line were purchased from ATCC (American

Type Culture Collection, Manassas, VA, USA). To facilitate the quantitative

measurement of tumor, a stable C6 cell clone with the firefly luciferase gene

(C6-Luc) was generated. NT2, BT325, U251, U87, H4, HepG2, and NIH3T3

were cultured in DMEM with fetal bovine serum (10%) and penicillin

streptomycin (1%). C6 cells were cultured in DMEM supplemented with 0.1

mM non-essential amino acids, fetal bovine serum (10%), and penicillin

streptomycin (1%). Complete growth medium supplemented with 0.1mg/ml

hygromycin were used for luciferase stable cell lines. All above mentioned cell

lines were cultured at 37ºC in a humidified incubator with 5%CO2. SW1783

and SW1088 were cultured in Leibovitz's L-15 medium with fetal bovine serum

(10%) and penicillin streptomycin (1%) at 37ºC in a humidified incubator with

100% air. Insect Sf9 cell line purchased from Invitrogen was cultured in Sf-900

II SFM medium with penicillin streptomycin (0.5%) at 27ºC in a non-humidified

incubator with 100% air.

Adult male Wistar rats (weighing 250–300 g) used for in vivo experiments

were obtained from Centre for Animal Resources in National University of

Singapore. During the handling and care of animals, we followed the

guidelines on the Care and Use of Animals for Scientific Purposes issued by


- 23 -

National Advisory Committee for Laboratory Animal Research, Singapore. The

experimental protocols of the current study were approved by the Institutional

Animal Care and Use Committee (IACUC), National University of Singapore

and Biological Resource Center, the Agency for Science, Technology and

Research (A* STAR), Singapore.

2.2 Shuttle plasmids and recombinant baculovirus production

We constructed nine recombinant baculoviral vectors (Table1) with

different expression cassettes based on the transfer vector pFastBac1

(Invitrogen, Carlsbad, CA, USA). Among two of them, a firefly luciferase

reporter gene (BV-CMV-Luc) or an enhanced green fluorescence protein

(eGFP) reporter gene (BV-CMV-eGFP) were under the control of the CMV

enhancer/promoter. GFAP promoter was used in three baculoviral vectors to

drive the expression of luciferase gene: the first one (BV-GFAP-Luc) has an

original GFAP promoter; in the second one (BV-CMV E/GFAP-Luc), a hybrid

GFAP promoter was generated by appending the CMV enhancer (-568 to -187

relative to the TATA box) to the 5’ end of GFAP promoter; in the third vector

(BV-CG/ITR-Luc), an expression cassette was constructed by flanking the

second cassette with AAV ITRs at both ends. In the other two vectors, the

luciferase gene in BV-CG/ITR-Luc was replaced by a DT-A gene

(BV-CG/ITR-DTA) or an eGFP gene (BV-CG/ITR-eGFP), respectively.


- 24 -

Table 1: Baculoviral vectors used in the current study Name Promoter Transgenes BV-CMV-Luc CMV Luciferase BV-CMV-eGFP CMV eGFP BV-GFAP-Luc GFAP Luciferase BV-CMV E/GFAP-Luc CMV E+GFAP Luciferase BV-CG/ITR-Luc CMV E+GFAP, ITR flanking Luciferase BV-CG/ITR-eGFP CMV E+GFAP, ITR flanking eGFP BV-CG/ITR-DTA CMV E+GFAP, ITR flanking DTA BV-H1-siLuc H1 Luciferase siRNA BV-CMV E/H1-siLuc CMVE+H1 Luciferase siRNA

To generate BV-CMV E/GFAP-Luc, a CMV enhancer sequence amplified

from pRC/CMV2 (Invitrogen, Carlsbad, CA, USA) was inserted into pFastBac1

between the sites of Not I and Xba I, and a GFAP promoter amplified from

pDRIVE02-GFAP (InvivoGen, San Diego, CA, USA) was subsequently

inserted downstream of the CMV E between Xba I and Xho I. To construct

BV-CG/ITR-Luc, an expression cassette from pAAV plasmid (Wang et al.,

2005), containing a multiple cloning site (MCS), a reporter gene encoding

luciferase, a SV40 polyA signal, and two ITR sequences at both ends, was

amplified and inserted into pFastBac1 between Avr II and Sal I. The CMV


- 25 -

E/GFAP promoter was then inserted into the sites of Kpn I and Hind III. The

BV-CG/ITR-eGFP and BV-CG/ITR-DTA were constructed by inserting an

eGFP reporter gene from peGFP-C1 vector (Clontech, Mountain View, CA,

USA), or a DT-A gene amplified from pCAG/DT-A-2 (kindly provided by Dr.

Masahiro Sato, Tokai University, Japan), respectively, into the downstream of

the GFAP promoter between the sites of Hind III and Xba I to replace the

luciferase gene. BV-CMV-Luc and BV-CMV-eGFP were constructed by

inserting the CMV promoter amplified from pRC/CMV2 into pFastBac1

between the Not I and Xba I and inserting between the sites of Xho I and Hind

III with a luciferase gene from pGL3-basic vector (Promega, Madison, WI, USA)

or eGFP gene from peGFP-C1 vector (Clontech, Mountain View, CA, USA),

respectively.

For the two vectors carrying siRNA genes, H1 promoter (BV-H1-siLuc) or

hybrid CMVE/H1 promoter was used in the expression cassettes of siRNA

targeting against luciferase. pRNAT-H1.1/Neo containing the human H1

promoter was purchased from GenScript (Piscataway, NJ, USA). To construct

the hybrid CMV E/H1 promoter, a CMV enhancer element (-568 to –87 relative

to the TATA box of the CMV immediate-early promoter) was amplified from

pRC/CMV2 (Invitrogen, Carlsbad, CA, USA) and subcloned into

pRNAT-H1.1/Neo at the 5’ region of the Pol III promoter between the sties of

Mlu I and Bgl II. Oligonucleotides

(5’-GCTTACGCTGAGTACTTCGATTCAAGAGATCGAAGTACTCAGCGTAAG


- 26 -

CTTTTT-3’) targeting against the firefly (Photinus pyralis) luciferase coding

region with cohesive BamH I and Hind III sites were chemically synthesized,

annealed and cloned into pRNAT-H1.1/Neo or pRNAT-H1.1/Neo with the CMV

enhancer. The two plasmid vectors were named pH1-siLuc and pCMV

E+H1-siLuc, respectively. To construct recombinant baculoviral vectors with

shRNA expression cassette, the firefly luciferase siRNA hairpin-loop sequence

under the H1 promoter or the hybrid CMV enhnacer/H1 promoter was

amplified from pH1-siLuc and pCMV E+H1-siLuc and subcloned into the

transfer vector pFastBac1. The two recombinant baculoviral vectors were

named BV-H1-siLuc and BV-CMV E/H1-siLuc, respectively.

Recombinant baculoviruses were produced and propagated in Sf9

insect cells according to the manual of the Bac-to-Bac baculovirus expression

system (Invitrogen, Carlsbad, CA, USA). To concentrate recombinant

baculoviruses, the clear supernatant was filtered with 0.45μm membrane,

centrifuged at 28,000×g for 1 hour at 4ºC and the pellet was suspended with

appropriate volume of 1X PBS by vortexing 30 minutes. The titers

(plaque-forming units, PFU) of the recombinant baculovirus vectors were

determined by plaque assay on Sf9 cells. The prepared baculovirus stocks

were stored at 4ºC and protected from light.


- 27 -

2.3 Virus transduction

For in vitro transduction, cells were seeded in 96-well plates at a density of

1,000 cells per well or 48-well plates at a density of 20,000 cells per well for

luciferase activity assay, in 12-well plates at a density of 100,000 cells per

well for flow cytometric analysis, in 96-well plates at a density of 10,000 cells

per well for MTT assay, in 6-well plate at a density of 100,000 cells per well for

RT-PCR analysis and in 24-well plates with a density of 30,000 cells per well

for gene silencing experiments. Cells were incubated with appropriate

amounts of baculoviral vectors in DMEM at 37°C for 1 hour. After the

incubation, DMEM containing the viruses was replaced by complete growth

medium and the infected cells were cultured in normal condition.

To characterize the gene expression profiles in vivo, C6 or C6-Luc cells (1

x 105 in 5 μl) were first implanted into the striatum on one side of the rat brain.

Three days later, 5 x 107 viral particles of BV-CG/ITR-eGFP or 5 x 106 of

BV-CG/ITR-Luc in 3 μl were injected into the same region, as well as the

contralateral striatum in some animals.

To test in vivo gene silencing effects, BV-H1-siLuc, BV-CMV E+H1-siLuc or

the control vector BV-CMV-eGFP, 9 x 107 virus particles each, were injected

together with BV-CMV-Luc (3 x 106 PFU per brain). Rats were euthanized 2

days after viral injection and the brain tissues were collected for gene

expression analysis.

To test the inhibition of tumor growth in vivo, 100,000 C6-Luc cells were


- 28 -

implanted into the striatum on both sides of the rat brain. Three days later, 1x

107 viral particles in 3μl were injected into the striatum at the same site.

BV-CG/ITR-DTA was injected into the left side and BV-CG/ITR-eGFP, serving

as a viral vector control, into the right side.

A standard operation protocol of the stereotaxic injection was followed.

Briefly, rats were anesthetized with intraperitoneal injection of sodium

phenobarbital (60mg/kg) and positioned in a stereotaxic instrument (KOPF,

Model 900, USA) with the nose bar set at 0. Then a skin incision of about 1 cm

in length was made in the appropriate position and the cranial bone was

exposed. A small hole was made in the skull by a dental drill according to the

stereotaxic anatomy atlas of rat brain. The cells or viruses were injected into

the striatum (AP+1.0 mm, ML +2.5 mm, and DV -5.0 mm from bregma and

dura) through the hole using a 10 μl Hamilton syringe connected with a

30-gauge needle at a speed of 0.5 μl/min. At the end of each injection, the

needle was allowed to remain in place for additional 5 minutes before being

slowly retracted to prevent the backflow.

2.4 Luciferase activity assay

To measure the luciferase expression in cultured cells, the growth medium

were carefully remove from the cells, and the cells were rinsed with 1X PBS

with care to avoid cell dislodging. Then cells were permeabilized by adding

appropriate volume of 1X reporter cell lysis buffer (Promega, WI, USA),


- 29 -

followed by two freeze/thaw cycles to further lyse the cells, and thus release

the luciferase. To measure the luciferase expression in brain tissues, rats were

perfused with 100ml 1X PBS after deep anesthesia. Brain tissue samples were

collected, homogenized by sonicator in 1X PBS (100 μl PBS per 50 mg tissue)

for 10 sec on ice, and centrifuged at 13,000 rpm for 10 minutes at 4°C. The cell

lysates or the supernatants of homogenized tissues were used for luciferase

activity assays with a luciferase assay kit (Luciferase Assay System, Promega,

WI, USA) in a single-tube luminometer (Berthold Lumat LB 9507, Bad Wildbad,

Germany). Ten μl of sample was mixed with 50μl of substrate from the

luciferase assay kit in a 10ml plastic tube. For in vitro study, the luciferase

activity was represented by relative light units (RLU) per 1000 cells. For the

luciferase expression in brain, the results were represented by RLU per region.

Luciferase activity in the protein synthesis inhibition experiment on C6-Luc

cell line and in the in vitro gene silencing experiment were monitored by BLI

with the IVIS® Imaging System (Xenogen, Alameda, California, USA)

comprised of a highly sensitive, cooled CCD camera mounted in a light-tight

specimen box. Two to five minutes prior to cell imaging, luciferin-EF (150 μg/ml

in 1X PBS; Promega, Madison, WI, USA) was added to each well.

Bioluminescence emitted from the cells was acquired for 30s and quantified as

photons/second using the Living Image software (Xenogen, Alameda,

California, USA). In some experiments, bioluminescence was digitized and

electronically displayed as pseudocolor.


- 30 -

2.5 Detection of eGFP expression

The expressions of eGFP in glioma cell lines were observed directly

with an inverted fluorescent microscope (Olympus IX71, USA). The

transduction efficiency was quantitatively measured by counting the

percentage of eGFP positive cells with flow cytometric analysis. For flow

cytometric analysis of eGFP expression, at certain time post transduction,

glioma cells were washed with 1X PBS, trypsinized, suspended in 1X PBS and

directly introduced to a FACSCalibur Flow Cytometer (Becton Dickinson, NJ,

USA) equipped with a 488 nm argon ion laser. The FL-1 emission channel was

used to monitor the eGFP expression and results from 10,000 fluorescent

events were obtained for analysis. Cells without virus transduction were served

as negative controls. Three sets of independent transduction experiments

were carried out for each assay.

2.6 RT-PCR

For detection of DTA expression, total RNA was extracted from U251 cells

transduced with BV-CG/ITR-DTA or BV-CG/ITR-eGFP using RNeasy® Mini

KIT (Qiagen, USA ) after on-column DNase digestion (RNase-Free DNase Set,

Qiagen, USA). RNA concentration was determined by spectrophotometer

(NanoDrop® ND 1000, USA). The DTA mRNA expression was determined

with SuperScript One-Step RT-PCR with Platinum® Taq kit (Invitrogen,

Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, 1μg of


- 31 -

the total RNA sample was used as the starting material for end-point RT-PCR

detection and analysis. Gene specific primers for the DTA gene were used,

Forward primer: 5’-AAATACGACGCTGCGGGAT-3’,

Reverse primer 5’-GAAGGGAAGGCTGAGCACTA-3’.

RT-PCR reaction was carried out in an Eppendorf® thermal cycle using

the following program: 50°C for 30 minutes and 94°C for 2 minutes, followed

by 38 cycles of 94°C for 15 sec, 58°C for 30 sec, and 72°C for 1 minutes, and

final extension at 72°C for 10 minutes.

For the detection of luciferase mRNA, 1 μg of total RNA from each sample

was used for the synthesis of first strand cDNA with SuperScript III First Strand

Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA). Oligo-dT was

used as primer. The cDNA was synthesized according to manufacturer’s

protocol. 1 μl of cDNA from each sample was used for PCR amplification. Each

reaction also contained 10 μl of TaqPCR Master Mix (Qiagen, USA), 1 μl each

of forward and reverse primers, and 7 μl of distilled water to make up a 20 μl

reaction volume. As a control to demonstrate equal amount of RNA used, each

sample was also amplified for the endogenous house-keeping gene, GADPH,

under the same conditions. For the PCR-detection of luciferase mRNA, the

primers used were

Forward primer 5’-CGAGGTGGACATCACTTACGCTG-3’,

Reverse primer 5’-CGAGAATCTCACGCAGGCAGTTC-3’.

The primers used for PCR amplification of GADPH were


- 32 -

Forward primer 5’-GAAGATGGTGATGGGATTTC-3’,

Reverse primer 5’- GAAGGTGAACGTCGGAGT-3’.

Aliquots of RT-PCR products were analyzed by gel electrophoresis on a

1% agarose gel containing ethidium bromide at a voltage of 80mV for

60minutes, and visualized by UV.

2.7 Fluorescence immunohistochemistry

Florescence immunohistochemistry was used to analyze cell type

specificity of transgene expression. After deep anesthesia, rats were perfused

with 100ml 1X PBS and 100ml 4% paraformaldehyde in 1X PBS. The brain

was taken out with care and incubated in the same fixative (4%

paraformaldehyde) for 2–4 hours before being transferred into 20% sucrose in

1X PBS for incubation overnight at 4°C. Coronal sections of each brain were

cut with cryostat (Leica CM3050S, USA). One piece of brain sample was put

on the cryostat stage, covered with OCT medium, and snap froze in liquid

nitrogen for 30 seconds. Then it was transferred to the cryostat chamber and

sectioned at thickness of 30µm. Sections of the regions of interest were

collected with care and transferred to a 6-well plate containing 1X PBS. A

free-floating immunostaining protocol was followed. Briefly, sections were

washed with washing solution (1X PBS containing 0.2% Triton X-100) for three

times 10minutes each, then blocked in blocking solution (1X PBS containing

0.2% Triton X-100 and 5% normal goat serum) for 1 hour at room temperature.


- 33 -

Primary antibody diluted in washing solution was added and incubated

overnight at 4 °C with gentle shaking. On the following day, sections were

washed with washing solution three times 10minutes each, secondary

antibody with a proper dilution was added and incubated at RT for 1 hour

preventing from light. After this incubation, sections were washed with 1X PBS

for five times 10minutes each. The sections were then carefully transfer onto a

gelatin-coated slide, cover with a drop of fluorescent mounting solution (DAKO,

USA), add cover slip, and store the slide at 4 °C overnight to allow mounting

medium to set. Sections were observed with a confocal laser scanning

microscope (Leica, TCS SP2 RS, USA). A anti-luciferase polyclonal antibody

(Sigma–Aldrich, USA, dilution 1:150) or a anti-GFAP polyclonal antibody

(Promega, USA, dilution 1:150) was used as the primary antibody to show

implanted C6 glioma cells, as well as nearby astrocyte, while the expression of

eGFP could be observed without fluorescence staining. Anti-rabbit IgG TRITC

conjugate (Sigma–Aldrich, USA; dilution 1:500) was used as the secondary

antibody.

2.8 Cell viability assay

Cells seeded in 96-well plates were transduced with BV-CG/ITR-DTA or

BV-CG/ITR-eGFP. Six days after virus transduction, 20 μl of 5 mg/ml MTT (3-(4,

5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) in 1X PBS was

added to each well to reach a final concentration of 0.5mg/ml. After 4 hours


- 34 -

incubation at 37°C, the medium was removed and 200 μl of DMSO was added

into each well to dissolve the formazan crystals. The absorbance was

measured in a microplate reader at a wavelength of 550 nm (BioRad, Module

550, USA). The relative cell growth (%) comparing with control cells without

virus infection was calculated as Atest/AcontrolX100%

2.9 Rat C6 glioma xenograft model and tumor growth monitoring

To establish a rat C6 tumor xenograft model, C6-Luc cells were trypsinized,

harvested by centrifugation, and suspended in 1X PBS. Then the cells

suspended in 5μl 1X PBS were implanted into the brain following the

stereotaxic injection protocol described in section 2.3. To measure the tumor

size, brain samples were taken at certain time points post implantation and

coronal sections with thickness of 0.3mm were cut out by cryostat (Leica

CM3050S, USA) as described above in section 2.7. β-gal staining (β-gal

staining kit, Invitrogen, Carlsbad, CA, USA) was used to visualize the C6-Luc

cells which also have the LacZ gene expression. The staining protocol

provided by the manufacture was followed with some optimization. The

incubation time of staining solution was increased to 4 hours. After staining,

sections were observed under microscope and the area of tumor was

measured. The total size of the tumor was calculated by following formulation:

Tumor size (mm3) =0.3 X (A1 + A2 + --- + An); in where An is the tumor area in

each section


- 35 -

Tumor growth was also monitored by either luciferase activity assay of

brain tissues or BLI of C6-Luc cells in living animals. To monitor glioma growth

with luciferase activity assay, brain samples were collected and the luciferase

activity was measured as described in section 2.4.

BLI was performed with the IVIS Imaging System (Xenogen, Alameda,

California, USA). Briefly, ten minutes before in vivo imaging, anesthetized

animals were intraperitoneally injected with D-luciferin (Promega, WI, USA) at

a concentration of 40 mg/kg in 1X PBS. The animals were then placed onto a

warmed stage inside the camera box. The detected light emitted from C6-Luc

cells was digitized and electronically displayed as a pseudocolor overlay onto

a gray scale animal image. Images and measurements of luminescent signals

were acquired and analyzed using the Living Image software (Xenogen,

Alameda, California, USA). Animals were euthanized 14 days after virus

injection.

Chapter Three: Results

- 36 -

Chapter Three

Results


- 37 -

3.1 Establishment of C6 glioma xenograft model

For the evaluation of gene therapy approaches, it is important to have a

reliable tumor model that can mimic the growth profiles of gliomas in vivo.

C6/Wistar rat xenograft model has been widely used in the studies of glioma

therapy (Barth et al., 1998; Zhang et al., 2002). In addition, the ease of

monitoring tumor growth and the tumor’s response to treatments is another

criterion for a good tumor model. In the current study, the C6 glioma cells have

been genetically engineered to have stable luciferase expression in order to

facilitate the monitoring of tumor by BLI or luciferase activity assay. As

indicated in Fig. 1, with increased amount of C6 cells implanted into the brain

(from 10,000 to 1,000,000), the tumor size increased correspondingly (from

4.2 to 39.9 mm3) and a similar increase profile was observed in the results

based on the luciferase activity assay (Fig. 2A). A good correlation between

the tumor size measurement and luciferase activity assay (Fig. 2B) was

demonstrated (R2 = 0.9998), which indicated that the luciferase activity assay

was a reliable method to monitor the glioma growth in vivo. Therefore, the

growth of gliomas over time could also be monitored by either luciferase

activity assay (Fig. 2C) or non-invasive BLI (Fig. 13).


- 38 -

A

B

0

10

20

30

40

50

60

1.00E+04 1.00E+05 1.00E+06

Implanted Cell Number

Tumor Size(mm^3)

Fig. 1 Monitoring the C6 glioma xenograft model by calculating the tumor size. Various number of C6-Luc cells (also with lacZ gene expression) were implanted into the rat brain with stereotaxic injection. One week after implantation, brain sections were treated with lacZ staining. (A) C6 glioma formed in the rat brain was visualized by lacZ staining. (B) Increase of tumor size with increased number of cells implanted. Tumor size was calculated by a serial section method. Columns, mean (n = 3); bars, SD.


- 39 -

A

1.00E+06

2.10E+07

4.10E+07

6.10E+07

8.10E+07

1.01E+08

1.00E+04 1.00E+05 1.00E+06Implanted Cell Number

RLU/Region

B

y = 2E+06x + 2E+06R2 = 0.9754

0.00E+001.00E+072.00E+073.00E+074.00E+075.00E+076.00E+077.00E+078.00E+079.00E+071.00E+08

0 10 20 30 40 50 60

Tumor Size (mm^3)

RLU

/Reg

ion

C

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

0 1 2 3 4

Time(week)

RLU/Region

Fig. 2 Monitoring the C6 glioma xenograft model by luciferase activity assay. (A) Various number of C6-Luc cells were implanted into the rat brain with stereotaxic injection. One week after implantation, brain samples were taken for luciferase activity assay. Luciferase activity increased with the increased number of cells implanted. Columns, mean (n = 3); bars, SD. (B) There was a good correlation between the tumor size and luciferase activity. (C) 100,000 C6-Luc cells were implanted into the rat brain with stereotaxic injection. One, two, and three weeks after implantation, brain samples were taken out for luciferase activity assay. Points, mean (n=3); bars, SD.


- 40 -

3.2 DTA expressing baculovirus-mediated inhibition of glioma cell

growth

3.2.1 Effective transduction of glioma cells by baculoviral vectors

Although recombinant baculovirus has been shown to be able to transduce

various types of mammalian cells (Kost et al., 2002), its ability to deliver

transgenes into glioma cells has not yet been well characterized. Therefore,

two baculoviral vectors with different reporter genes, namely BV-CMV-Luc

(luciferase gene) and BV-CMV-eGFP (eGFP gene), under the control of the

CMV promoter were first used to test the baculovirus-mediated gene delivery

in seven glioma cell lines, including C6, H4, SW1088, SW1783, U87, U251

and BT325. The ubiquitous transcriptional activity of the strong CMV promoter

enabled the comparison of the gene expression levels between these glioma

cell lines with different grades of malignancy. As indicated in Fig. 3,

BV-CMV-Luc was able to achieve comparable levels of luciferase expression

in all the tested gliomas cell lines in a dose-dependent pattern, though with a

slightly higher level in U87 and lower level in SW 1783 at a multiplicity of

infection (MOI) of 100 or 200. For BV-CMV-eGFP, the eGFP expression in

glioma cells was observed as early as 4 to 6 hours post transduction. The flow

cytometric analysis of these cells 24 hours post transduction showed a

dose-dependent increase of the percentage of eGFP positive cells, ranging

from 30% to 70% at a MOI of 100 (Fig. 4). Further increase of the MOI to 200

achieved only 10% improvement, indicating a plateau of the transduction


- 41 -

efficiency in glioma cells was reached at the MOI of around 100.

Fig. 3 Transduction of glioma cells with baculovirus with luciferase reporter gene. Cells were transduced with BV-CMV-Luc with increased MOI from 1 to 100. Luciferase activity assay was carried out one day after infection. The results are expressed in relative light units (RLU) per 1000 cells. Points, mean (n=4); bars, SD

1000

10 5

10 7

C6SW1088 SW1783 H4U87 U251 BT325

MOI

Lucif

eras

e Act

ivity

(RLU

per

1000

cells

)

0 20 40 60 80 100


- 42 -

Fig. 4 Transduction of glioma cells with baculovirus with eGFP reporter gene. Cells were transduced with BV-CMV-eGFP with increased MOI from 10 to 200 and analyzed with flow cytometry one day later. The results are reported as the percentage of eGFP-positive cells. Points, mean (n=4); bars, SD.

0

40

80

% o

f eGF

P po

sitive

cells

MOI

C6 SW1088

SW1783 H4

U87 U251 BT325

200 50 0 100 150


- 43 -

3.2.2 Modified GFAP promoters improve transgene expression to glioma

cells

The use of cell type-specific promoter is an important strategy to drive the

expression of therapeutic genes within targeted cells, while reducing adverse

effects caused by over-expression of therapeutic genes in non-target cells. In

order to restrict the gene expression in glioma cells, we constructed a

baculoviral vector, BV-GFAP-Luc, in which a luciferase expression was under

the control of a GFAP promoter (Fig. 5A). However, only a low level of

luciferase expression was achieved in the tested glioma cells, being 10 to

several hundred-fold lower than those from the baculoviral vector containing

the CMV promoter (BV-CMV-Luc; Fig. 5B). To enhance the transcriptional

activity of the GFAP promoter, two additional transcriptional regulatory

elements were incorporated into the expression cassette of the baculoviral

vector (Fig. 5A). In one of the modification (BV-CMV E/GFAP-Luc), we inserted

the CMV enhancer from cytomegalovirus upstream to the GFAP promoter; in

another one (BV-CG/ITR-Luc), we flanked the expression cassette (hybrid

CMV E/GFAP promoter and luciferase transgene) with the ITR sequences

from adeno-associated virus. As indicated in luciferase activity assay (Fig. 5B),

the CMV enhancer greatly increased the transcriptional activity of the GFAP

promoter in all tested glioma cell lines, leading to high levels of luciferase

expression comparable to those achieved with baculovirus containing the


- 44 -

strong CMV promoter. The AAV ITR flanking further increased the luciferase

expression by at least 10 folds compared with BV-CMV E/GFAP-Luc. In 5 out

of 7 tested glioma cell lines, even higher levels of luciferase expression were

achieved from BV-CG/ITR-Luc compared with BV-CMV-Luc (Fig. 5B). It was

also observed that although BV-CMV-Luc provided similar levels of luciferase

expression in two non-glioma cell lines, namely HepG2 and NIH3T3,

BV-GFAP-Luc, BV-CMV E/GFAP-Luc and BV-CG/ITR-Luc, in contrast, showed

significant lower levels of luciferase expression in HepG2 and NIH3T3 cells,

when compared to those in glioma cell lines (Fig. 5B). Since similar levels of

transgene expression in the two non-glioma cell lines and those glioma cell

lines were achieved by BV-CMV-Luc, the difference in cellular uptake and

intracellular transport of baculovirus per se should not be the reason for the

low levels of transgene expression in HepG2 and NIH3T3 cells. These results

indicated that our modification of the GFAP promoter was able to enhance the

transcriptional activity, while retaining good cell type specificity.

Although the luciferase activity assay is a sensitive quantitative method for

comparison of gene expression levels, especially in the study to compare the

transcriptional activity of different gene expression cassettes, the eGFP

reporter gene can provide direct information regarding the transduction

efficiency based on the percentage of eGFP positive cells. For this purpose, a

baculovirus with hybrid CMV E/GFAP promoter flanked by AAV ITR sequences

to drive the expression of the eGFP gene (BV-CG/ITR-eGFP) was constructed


- 45 -

by replacing the luciferase gene with the gene encoding eGFP (Fig. 6A). When

this baculovirus was tested, a high level of eGFP expression in glioma cell

lines was observed with the inverted florescence microscope (Fig. 6C). Flow

cytometric analysis indicated a significant improvement in transduction

efficiency over the those achieved from baculovirus with CMV promoter

(BV-CMV-eGFP) in all the tested glioma cell lines, with percentage of eGFP

positive cells ranging from 54% in SW1088 to 98% in C6 cells (Fig. 6B).


- 46 -

A

B

Fig. 5 Modified GFAP promoters improved baculovirus-mediated luciferase expression in glioma cells. (A) Schematic diagram of the expression cassettes used in the study. BV, baculovirus; CMV, the promoter/enhancer of cytomegalovirus immediate-early gene; GFAP, the promoter of the glial fibrillary acidic protein; CMV E, the enhancer of cytomegalovirus immediate-early gene; ITR, AAV inverted terminal repeats; Luc, luciferase gene; pA, SV40 polyA signal. (B) Cells were infected with the baculoviral vectors with a luciferase reporter gene under the control of different expression cassettes at an MOI of 25. Luciferase activity assay was performed one day after transduction. Results are reported as the percentage of RLU produced by the vector with the CMV promoter. Columns, mean (n = 4); bars, SD.

BV-CMV-Luc BV-GFAP-Luc BV-CMV E/GFAP-Luc

BV-CG/ITR-Luc CMV E/GFAP luc pA ITR ITR

CMV E/GFAP luc pA

GFAP luc pA

CMV luc pA

0

200

400

600

U251

U87

BT32

5 H4

SW10

88

SW17

83

C6

HepG

2

NIH3

T3

(% o

f CMV

Pro

mot

er )

Lucif

eras

e Act

ivity


- 47 -

A

B

Fig. 6 Modified GFAP promoters improved baculovirus-mediated eGFP expression in glioma cells. (A) Schematic diagram of the expression cassettes used in this study. BV, baculovirus; CMV, the promoter/enhancer of cytomegalovirus immediate-early gene; GFAP, the promoter of the glial fibrillary acidic protein; CMV E, the enhancer of cytomegalovirus immediate-early gene; ITR, AAV inverted terminal repeats; eGFP, enhance green fluorescence gene; pA, SV40 polyA signal. (B) Cells were infected with the baculoviral vectors with two different expression cassettes at an MOI of 100 and analyzed with flow cytometry one day later. The results are reported as the percentage of eGFP-positive cells. The results from the experiment with BV-CMV-eGFP in Fig. 4 are included for comparison. Columns, mean (n = 4); bars, SD.

0

40

80

U251

U87

BT32

5

H4

SW10

88

SW17

83

C6

BV-CMV-eGFPBV-CG/ITR-eGFP

% o

f EGF

P po

sitive

cells


- 48 -

Fig. 6 Modified GFAP promoters improved baculovirus-mediated eGFP expression in glioma cells (C) Cells were infected with BV-CG/ITR-eGFP at an MOI of 100 and pictures were taken with digital camera attached to Olympus IX71 inverted fluorescence microscope one day after transduction.


- 49 -

3.2.3 Inhibition of protein synthesis and glioma cell growth in vitro

In order to explore the feasibility of using baculovirus as a vector for glioma

gene therapy, a recombinant baculovirus (BV-CG/ITR-DTA) was constructed

by replacing the luciferase reporter genes in BV-CG/ITR-Luc with the gene

encoding diphtheria toxin A-chain. In this baculoviral vector, the DTA gene

expression was under the control of an expression cassette composed of

hybrid CMV E/GFAP promoter and flanked by AAV ITRs. Owing to the tight

transcriptional control of this GFAP promoter based expression cassette in the

insect cells, high titer viral preparation were successfully produced in Sf9

insect cells despite the high toxicity of DTA. We first confirmed this baculoviral

vector (BV-CG/ITR-DTA) mediated DTA expression in U251 glioma cells by

RT-PCR using DTA gene specific primers (Fig. 7). Then the inhibition of protein

synthesis in glioma cell lines by BV-CG/ITR-DTA was evaluated according to a

method first reported by Maxwell (Maxwell et al.,1986), which is an indirect

method based on the effects of DTA on co-expressed luciferase protein. Six

glioma cell lines, namely H4, SW1088, SW1783, U87, U251 and BT325, were

co-transduced with BV-CMV-Luc and BV-CG/ITR-DTA or BV-CMV-Luc and

BV-CG/ITR-eGFP. The luciferase activity was measured 48 hours post

transduction.


- 50 -

Fig. 7 RT-PCR analysis of DTA expression. U251 cells were infected BV-CG/ITR-DTA at an MOI of 100. 48 hours after transduction, total RNA was extracted for RT-PCR analysis with DTA gene specific primers. Down arrow: A clear PCR band with a predicated size of 250. BV-CG/ITR-eGFP was served as a negative control.

As demonstrated in Fig. 8, a significant reduction of the luciferase activity

was observed in all the tested glioma cell lines transduced with the

BV-CG/ITR-DTA, even at a low MOI of 10, varying from around 50% inhibition

in BT325 to almost 90% inhibition in SW1088 cells. Although a slightly

reduction of the luciferase activity after transduction with control virus

(BV-CG/ITR-eGFP) was observed in some tested glioma cell lines, the more

dramatic effects were obvious when the viruses expressing DTA

(BV-CG/ITR-DTA) were used.

For the evaluation of DTA inhibition effects over time, BLI with the IVIS®

Imaging System was used to continuously monitor the temporal change of


- 51 -

luciferase activity over 6 days in C6-Luc cells, which were genetically modified

to stably express the luciferase gene. As demonstrated in Fig. 9,

BV-CG/ITR-DTA transduction resulted in an obvious reduction of luciferase

activity in C6-Luc cells, from 59% of the control on day 2 to 32% on day 6 at an

MOI of 50, and from 38% of the control on day 2 to 14% by day 6 at an MOI of

100. Inhibition of protein synthesis might eventually lead to cell death, which, in

turn, would be another reason for the reduction of luciferase activity on day 6.

At the MOI of 10, from 20% to 30 % inhibition was observed on day 2 and 3 but

not at later time points, this is probably due to rapid proliferation of

untransduced C6-Luc cells (Fig. 9). Cell viability assay was carried out to

evaluate the effects of BV-CG/ITR-DTA on cell growth directly. The inhibition of

cell growth was tested in two glioma cell lines (C6-Luc and U87), as well as in

two non-glioma cell lines (HepG2 and NIH3T3). These cells were transduced

with BV-CG/ITR-DTA or BV-CG/ITR-eGFP at an MOI of 100, and the MTT

assay was performed 6 days post transduction. As indicated in the cell viability

results (Fig.10), transduction of BV-CG/ITR-DTA led to 90% of growth inhibition

in C6-Luc cells and 40% in U87 glioma cells, but had no obvious effects in

HepG2 and NIH3T3 cells. However, no significant inhibition of cell growth was

observed in the four cell lines transduced with BV-CG/ITR-eGFP.


- 52 -

Fig. 8 BV-CG/ITR-DTA mediated inhibition of protein synthesis in cultured

glioma cell lines. Protein synthesis inhibition, as demonstrated by the reduction of luciferase activity, was measured 48 hours after transduction with increased MOI from 10 to 100 in six glioma cell lines. Points, mean (n=4); bars, SD.

BV-CG/ITR-eGFP BV-CG/ITR-DTA

0

40

80

120

0 20 40 60 80 100

U251RL

U (

% o

f con

trol)

0

40

80

120

0 20 40 60 80 100

H4

0

40

80

120

0 20 40 60 80 100

U87

RLU

( %

of c

ontro

l)

0

40

80

120

0 20 40 60 80 100

SW1088

0

40

80

120

0 20 40 60 80 100

BT325

RLU

( %

of c

ontro

l)

0

40

80

120

0 20 40 60 80 100

SW1783

MOI

MOI

MOI


- 53 -

Fig. 9 BV-CG/ITR-DTA mediated inhibition of protein synthesis in C6-Luc cell

line. After transduction of BV-CG/ITR-DTA in C6-Luc cells, time-dependent effects over 6 days were examined using the IVIS imaging system. Columns, mean (n = 4); bars, SD.

80

MOI 0

MOI 10

MOI 50

MOI 100

120

80

40

0

C6-Luc

Phot

ons p

er se

c (%

of c

ontro

l)

2 3 4 5 6 Days


- 54 -

Fig. 10 BV-CG/ITR-DTA mediated selective inhibition of glioma cell growth in vitro. Cells were transduced with BV-CG/ITR-DTA or BV-CG/ITR-eGFP (as control) at an MOI of 100. Six days after baculovirus transduction, cell viability was determined by MTT assay. Columns, mean (n = 4); bars, SD.

0

40

80

120

U87 C6 HepG2 NIH3T3

BV-CG/ITR-eGFP BV-CG/ITR-DTA

% o

f cell

viab

ility


- 55 -

3.2.4 Expression of reporter genes in glioma xenograft

In order to further investigate the use of recombinant baculovirus for

glioma therapy in vivo, the baculovirus-mediated expression of two reporter

genes encoding eGFP or luciferase were evaluated in the C6 glioma xenograft

model. In the first study, C6-Luc cells were implanted into the rat striatum and 3

days later BV-CG/ITR-eGFP was injected into the glioma at the same position.

Two days after baculovirus injection, the brain sample was collected and an

immunohistochemistry study was carried out. The eGFP expression was

observed in luciferase-positive C6 cells (Fig. 11). In the sections stained with

antibodies against GFAP, the reactive gliosis was observed, marking the

boundary of solid glioma by a rim of reactive astrocytes with strong GFAP

signal. High level eGFP expression was also observed in many of these

reactive astrocytes (Fig. 11, the right panel). However, no detectable eGFP

expression was observed in the normal tissues outside the gliosis rim.


- 56 -

Fig. 11 In vivo eGFP reporter gene expression in gliomas mediated by baculovirus carrying the hybrid CMV E/GFAP promoter and ITRs. BV-CG/ITR-eGFP was injected into the rat striatum that was inoculated with C6-Luc glioma cells 3 days before. Immunostaining was carried out to show C6 cells and nearby astrocytes, while eGFP expression could be visually detected under a fluorescent microscope without immunostaining. The left panel, immunostaining with antibody against luciferase show glioma tissues. The right panel, immunostaining with antibody against GFAP show reactive gliosis surrounding the inoculated tumor cells (T).


- 57 -

To compare the in vivo transgene expression levels in glioma cells and

normal astrocytes, a recombinant baculovirus with luciferase gene

(BV-CG/ITR-Luc) was used. C6 glioma cells without modification were

implanted into one side of the rat striatum three days before virus injection.

Then same amount of BV-CG/ITR-Luc were injected into the C6

cells-inoculated brain region and the contralateral side of the rat brain without

C6 inoculation, respectively. Two days post virus injection, the brain tissues

were collected and the luciferase activity was measured. As shown in Fig. 12,

luciferase expression level in C6-inoculated brain region was 10 folds higher

than that in normal brain, which might be due to the higher transcriptional

activity of BV-CG/ITR-Luc expression cassette in glioma cells with the reactive

astrocytosis.

Fig. 12 In vivo luciferase gene expression in gliomas mediated by baculovirus carrying the hybrid CMV E/GFAP promoter and ITRs. BV-CG/ITR-Luc was injected into the rat striatum inoculated with C6 glioma cells (without the luciferase gene) 3 days before, and the contralateral normal striatum. Luciferase expression was measured 2 days after the virus injection by luciferase activity assay. The results are expressed in relative light units (RLU) per brain. Columns, mean (n = 4); bars, SD.

10 4

10 5

10 6

Normal Glioma

RLU

per

regi

on


- 58 -

3.2.5 Inhibition of glioma xenograft growth

After showed the BV-CG/ITR-DTA mediated effective inhibition of glioma

cell growth in vitro and the efficient transfer of reporter genes into the glioma

cells in vivo with recombinant baculovirus with same expression cassette, we

further test the anti-glioma effects of BV-CG/ITR-DTA in vivo in a C6 glioma

xenograft model. C6-Luc cells were implanted into the striatum at both

hemispheres of the brain. Three days later, we injected BV-CG/ITR-DTA into

the left striatum and BV-CG/ITR-eGFP as the control into the right striatum at

the same site as those for glioma cell implantation. BLI with IVIS imaging

system was used to non-invasively monitor the glioma tumor growth in living

animals on 0, 3, 7 and 14 days after virus injection. The bioluminescence

signals from the implanted C6-Luc cells on the control side were detectable

from day 3 and the intensity of bioluminescence signals increased

continuously during the 14-day experiment. In contrast, on the

BV-CG/ITR-DTA injected side of the same rat, no detectable bioluminescence

signals were observed (Fig. 14A). Quantitative results from these rats are

summarized in Fig. 14B, indicating a significant inhibition of C6 glioma cell

growth in vivo by just one injection of BV-CG/ITR-DTA. We also performed

luciferase activity assay to monitor the glioma tumor growth in the brain. Brain

tissue samples from both sides of C6-Luc implanted rats were collected at day

0 (3 days post C6 cell implantation) and day 14 post the baculovirus injection.

Before the virus injection (day 0), there is no obvious difference in luciferase


- 59 -

activities from two sides of the brain. But after two weeks of viral injection, a

30-fold higher luciferase activity was observed on the BV-CG/ITR-eGFP

injected side, when compared with that in BV-CG/ITR-DTA-injected side (Fig.

13), which indicated that the DTA expression mediated by BV-CG/ITR-DTA

effectively inhibited the growth of glioma xenograft in the brain.

Fig.13 Monitoring the C6 glioma xenograft growth in the rat brain by luciferase activity assay. Rats were inoculated with C6-Luc cells to each side of the brain, followed by injection of BV-CG/ITR-DTA on the left side and BV-CG/ITR-eGFP on the right side 3 days later (designated as day 0). Measurement of tumor growth by luciferase activity assays of brain tissues collected at day 0 (n=3) and day 14 (n=5). The results are expressed in relative light units (RLU) per brain and presented as means with SD.

106

107

108

109

C6+BV-CG/ITR-DTAC6+BV-CG/ITR-eGFP

RLU

per

regi

on

Day 0 Day 14


- 60 -

A

B

Fig. 14 Monitoring the C6 glioma xenograft growth in the rat brain by BLI. Rats were inoculated with C6-Luc cells to each side of the brain, followed by injection of BV-CG/ITR-DTA on the left side and BV-CG/ITR-eGFP on the right side 3 days later (designated as day 0). (A) In vivo bioluminescent images of the brains with inoculated C6-Luc cells 3, 7 and 14 days after virus injection in a living animal. The luminescent light emitted from the side injected with the control viruses was easily detected and increased over time. No light could be detected on the BV-CG/ITR-DTA injected side. (B) Quantification of in vivo bioluminescence. Point, mean photon counts over time; bars, SD. Photon counts at day 0 were not much different form background bioluminescence. n = 6 at days 3 and 7 and n = 5 at day 14, as one rat died at day 12.

0

1 106

2 106

3 106

4 106

C6+BV-CG/ITR-DTAC6+BV-CG/ITR-eGFP

Mea

n ph

oton

cou

nts

10 5 15 Days


- 61 -

3.3 siRNA expressing baculovirus-mediated gene silencing

3.3.1 Knockdown of luciferase gene expression in cultured cells

To explore the feasibility of using baculovirus for delivery and expression of

siRNA, BV-H1-siLuc with an shRNA against the firefly luciferase reporter gene

and BV-CMV-Luc with the luciferase reporter gene, were first used for

co-transduction of human NT2 cells at a viral MOI of 200. Co-transduction of

BV-CMV-eGFP and BV-CMV-Luc was served as a control to indicate

transduction efficiency. RT-PCR analysis demonstrated that the luciferase

mRNA decreased upon the infection of BV-H1-siLuc (Fig. 15A). We used BLI

to continuously monitor the luciferase expression on infected NT2 cells at 24

and 48 hours after transduction. As indicated in Fig. 15B, BV-H1-siLuc

significantly inhibited expression of luciferase from BV-CMV-Luc at both time

points.

We further evaluated whether the incorporation of CMV enhancer in the

expression cassette will improve the siRNA expression controlled by H1

promoter and thus enhance the silencing effects (Fig. 16 & Fig. 17). NT2 and

C6 cells seeded in a 24-well plate were infected with BV-CMV-Luc at an MOI of

200 and allowed to express the luciferase report gene without interference for

one day. The cells were then infected again with BV-H1-siLuc, BV-CMV

E/H1-siLuc or BV-CMV-eGFP (as vector control) at the same MOI. The

luciferase expression on the same wells were measured daily by BLI with the

IVIS® imaging system. It was demonstrated that BV-H1-siLuc was able to


- 62 -

inhibit luciferase expression from BV-CMV-Luc in C6 cells at day 3 and in NT2

cells at both day 2 and 3. The maximum inhibition at day 3 was 80% in C6 cells

and 60% in NT2 cells relative to the viral vector control. After the incorporation

of the CMV enhancer into the expression cassette, the gene silencing effects

of the baculoviral vectors were enhanced. Firstly, the inhibition became

effective earlier. While BV-H1-siLuc infection did not result in statistically

significant decrease in luciferase expression in C6 cells at day 2, BV-CMV

E/H1-siLuc infection led to 80% of inhibition in this cell line at the same time

point. Secondly, the inhibition was more effective. At day 3, the inhibition

increased statistically significantly from 80% provided by BV-H1-siLuc to 95%

by BV-CMV E/H1-siLuc in C6 cells and 60% to 80% in NT2 cells, respectively.


- 63 -

A

B Fig. 15 Baculovirus-mediated gene silencing effects in vitro. NT2 cells (3 x 104 cells per well in a 24-well plate) were co-infected with BV-CMV-Luc and BV-H1-siLuc or BV-CMV-eGFP (as a vector control), at an MOI of 200 each. (A) RT-PCR analysis. Two days after viral infection, cells were collected to extract total RNA. RT-PCR was carried out using specific primers for the luciferase gene and GAPDH gene. (B) Monitoring of luciferase expression in living NT2 cells with BLI. Bioluminescence signals were captured 24 and 48 h after infection.


- 64 -

A

B Fig.16 Quantitative analyses of baculovirus-mediated gene silencing effects in C6 cells. Cells were seeded at a density of 3 x 104 cells per well in a 24-well plate and infected with BV-CMV-Luc at a viral MOI of 200. Twenty-four hours after the first infection, the cells were infected with BV-H1-siLuc, BV-CMV E/H1-siLuc or BV-CMV-eGFP, at an MOI of 200. (A) Luciferase activities are expressed as photons per sec over time. Points, mean (n=4); bars, SD. (B) The data were first normalized with pre-infection samples and calculated as percentage of bioluminescence signals of test samples against control samples over time. Columns, mean (n = 4); bars, SD. +++, p<0.001 compared with BV control; ** p<0.01 and *** p<0.001, compared with BV-H1-siLuc.

10 5

10 6

10 7

1 2 3

C6

BV-H1-siLuc BV-CMV E/H1-siLuc

Phot

ons

per s

ec

0

100

200

1 2 3

BV-CMV E/H1-siLuc

% o

f BV

Con

trol

+++

*** +++

BV-H1-siLuc

BV control

Days

Days +++

**

BV control


- 65 -

A

B Fig. 17 Quantitative analyses of baculovirus-mediated gene silencing effects in NT2 cells. Cells were seeded at a density of 3 x 104 cells per well in a 24-well plate and infected with BV-CMV-Luc at a viral MOI of 200. Twenty-four hours after the first infection, the cells were infected with BV-H1-siLuc, BV-CMV E/H1-siLuc or BV-CMV-eGFP, at an MOI of 200. (A) Luciferase activities are expressed as photons per sec over time. Points, mean (n=4); bars, SD. (B) The data were first normalized with pre-infection samples and calculated as percentage of bioluminescence signals of test samples against control samples over time. Columns, mean (n = 4); bars, SD. +++, p<0.001 compared with BV control; ** p<0.01 and *** p<0.001, compared with BV-H1-siLuc.

10 6

10

1 2 3

NT2

Phot

ons

per s

ec

BV control

BV-CMV E/H1-siLuc

6

5

BV-H1-siLuc

+++ **

+++

++++++

0

40

80

1 2 3

% o

f BV

Con

trol

BV control

BV-CMV E/H1-siLucBV-H1-siLuc

120

+++

Days

Days

+++

**


- 66 -

3.3.2 Knockdown of luciferase gene expression in rat brain

Given the effective knockdown of the target gene in vitro, we next tested

whether baculovirus-mediated siRNA expression was able to silence the target

gene expression in vivo. We co-injected BV-CMV-Luc together with

BV-H1-siLuc, BV-CMV E/H1-siLuc or the control vector BV-CMV-eGFP into the

rat striatum. Two days after injection, rat brains were taken and dissected into

three parts, the striatum, the cerebral cortex and the rest of the brain.

Luciferase expression in each brain region was measured separately by

luciferase activity assay. In the striatum, 60% reduction of the luciferase

expression was achieved by BV-H1-siLuc and 82% reduction by BV-CMV

E/H1-siLuc (Fig. 18). When results from the three parts of the brain were

combined together, the overall reduction of luciferase expression was 50 and

80% by BV-H1-siLuc and BV-CMV E/H1-siLuc, respectively (Fig. 18). The

significant difference between the inhibitions induced by the two viral vectors

indicated that, the in vivo gene silencing effects provided by the H1 construct

could be improved by the CMV enhancer. These results are promising, in view

of a recent study reporting the failure to induce RNA interference by direct

injection of synthetic siRNA into the brain, despite the RNA silence effects were

demonstrated in vitro (Isacson et al., 2003).


- 67 -

0

4 10 6

8 10

1.2 10 7

1.6 10 7

Striatum R

LU p

er s

tria

tum

+

0

2 10

4 10

6 10 Cerebral cortex

RLU

per

cer

ebra

l cor

tex

+

0

5 10

1 10

1.5 10

2 10

BV

cont

rol

BV-

H1

siLu

c

BV-

CM

V E

/H1-

siLu

c

Whole brain

RLU

per

bra

in

+++

+

+++

+

+

+

*

6

6

6

6

6

6

7

7

7

FIG.18. Baculovirus mediated- silencing effects in rat brain. Two days after co-injection of BV-CMV-Luc and BV-H1-siLuc, BV-CMVE+H1-siLuc or BV-CMV-eGFP (as a vector control), tissue samples were collected for luciferase activity assay. Four brain samples were used for each group. Values are expressed as RLU per tissue. +, p<0.05 and +++, P<0.001, respectively, compared with the control viral vector. *, p<0.05 compared with BV-H1-siLuc.

Chapter Four: Discussion and Conclusion

- 68 -

Chapter Four Discussion and Conclusion


- 69 -

As a promising gene for cancer therapy, the DTA gene has shown its

efficacy in many studies (Maxwell et al., 1986; Massuda et al., 1997; Li et al.,

2002; Ohana et al., 2002; Peng et al., 2002). This bacterial protein encoded by

the DTA gene is highly toxic when introduced into the cytoplasm of eukaryotic

cells. It inhibits protein synthesis by catalyzing ADP ribosylation of the

diphthamide group of cellular elongation factor 2, and kills cells through an

apoptosis pathway (Michl et al., 2004). Although so-called suicide gene

therapy could also been accomplished by using other genes, such as HSV-tk

followed by systemic administration of the prodrug GCV (Izquierdo et al., 1996),

the efficacy of HSV-tk/GCV approach is often undermined by the blood brain

barrier which make it difficult to achieve sufficient GCV concentration within the

gliomas located in the brain. However, for DTA-based gene therapy, only

minimal DTA expression is required to induce cell death in a cell cycle

independent way, killing both dividing and non-dividing tumor cells (Rodriguez

et al., 1998).

Due to the high toxicity of DTA in mammalian cells, its accurate targeting

to tumor cells is necessary in order to avoid unintended deleterious effects on

non-target cells, especially for the treatment of gliomas located in the

vulnerable CNS. Glioma-specific gene expression could be realized by two

strategies. The first one is to utilize the natural tropism of a delivery vector or to

retarget the vectors by conjugating with glioma-specific ligand molecules (Van

et al., 2002). The results of the current study have demonstrated that


- 70 -

baculovirus was able to effectively transduce glioma cell lines in vitro (Fig. 1)

and, in addition, the tropism of baculovirus towards glial cells, not the neuron

cells, in brain was observed in previous studies (Sarkis et al., 2000; Li et al.,

2004). However, the tropism of vector per se is usually not enough and many

factors will affect the targeting accuracy. Therefore, a tissue- or tumor-specific

promoter has been used as another approach to ensure glioma targeting at the

transcriptional level (Shinoura et al., 2000; Miyao et al., 1993). In the current

study, a GFAP promoter was used to restrict the expression of DTA in glioma

cells. In combination with the intra-tumor injection procedure, the use of the

cell type-specific promoter could significantly reduce the risk of disturbing

neuronal functions caused by the over-expression of the toxin gene. Although

the expression of transgenes in astroglia , surrounding the boundary of the

glioma might occur, this is less likely to cause severe adverse effects to the

CNS, because the astroglia are not crucial for normal CNS function.

Compared with the ubiquitous viral promoter, one major disadvantage of

cellular promoters is their weak transcriptional activity, which results in low

levels of transgene expression and affects their therapeutic efficacy. In order to

overcome this problem, we modified the GFAP promoter by incorporating

additional transcriptional regulatory elements. In the context of a baculoviral

vector, the transcriptional activity of the GFAP promoter was enhanced by

incorporating a CMV enhancer, and was further improved by adding AAV ITRs.

We also demonstrated that the glial cell specificity of the GFAP promoter was


- 71 -

not impaired by this modification. The GFAP promoter is inactive in insect cells,

as revealed by our observations that while a baculovirus with an eGFP gene

driven by the viral CMV promoter produced green fluorescence in insect cells,

the baculovirus using the GFAP promoter did not show any eGFP-positive

insect cells. This cellular specificity has made it possible for us to produce high

titer recombinant baculovirus containing the DTA gene. We have thus

demonstrated the usefulness of the baculovirus/insect cell system to generate

recombinant toxin gene-expressing viral vectors suitable for targeted toxin

gene expression in tumor cells. The low levels of luciferase expression in two

tested non-glioma cell lines and their resistance to BV-CG/ITR-DTA

transduction further proved the glial cell specificity of this GFAP

promoter-based expression cassette. Interestingly, our recombinant

baculovirus accommodating the engineered expression cassette could

achieve a higher level of transgene expression in the glioma cell-inoculated

brain region than in the normal brain region. This could be explained by the

active proliferation of tumor cells and/or gliosis in the surrounding regions in

response to the damage caused by glioma invasion. The baculoviral vector

created in the current study is a promising vector for glioma gene therapy and

its performance in the treatment of gliomas will be further improved if an

authentic glioma-specific promoter is employed to further increase the

specificity and efficiency of this baculoviral vector.

A rat C6 xenograft animal model was used in the current study, allowing


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the non-invasive monitoring of tumor growth in vivo. High transduction

efficiency (98%) was observed with C6 cells in vitro, when baculovirus with the

CG/ITR expression cassette was used. Based on the high transduction

efficiency and the potent effect of DTA, it is reasonable to observe a significant

inhibition of C6 cell growth in animal model (Fig. 3C & D). The failure to

eliminate more C6 cells in the in vivo study is mainly due to the poor diffusion

of injected baculovirus within the tumor mass. Therefore, it is possible to

further improve the inhibition of C6 glioma growth by multiple injections of

viruses into several sites of the glioma tumor.

The success of RNAi-based gene therapy also relies greatly on the

development of vectors, especially on the choice of expression cassette that

controls shRNA expression. Pol III promoters are distinguished by their high

transcriptional activity, which is able to provide approximately 4 x 105

transcripts per cell (Matthess et al., 2005). However, in certain circumstances,

the activities of the original Pol III promoters are not sufficient to provide

satisfactory gene silencing outcomes (Ilves et al., 1996; Boden et al., 2003;

Thompson et al., 1995; Paul et al., 2002; Xia et al., 2003). To enhance the

transcriptional activity and improve the knockdown of target gene expression,

a hybrid promoter was created in the current study by fusing the CMV

enhancer 5’ to the H1 promoter. Our findings are consistent with those from a

previous study in which the CMV enhancer was fused to the U6 promoter (Xia

et al., 2003). Both the H1 and U6 promoters are type III Pol III promoters and


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are located only in the 5’-flanking region of the genes transcribed by them. This

gene-external feature resembles that of Pol II promoters but differs

significantly from that of types I and II of Pol III promoters, which are located

entirely within the transcribed region. While the Pol III U6 promoter directs the

synthesis of the U6 snRNA, Pol II promoters are used for transcription of the

U1 to U5 snRNAs. Strikingly, not only is the spatial organization of the U6

promoter elements similar to that of the U2 elements, they also share

homologous sequence in the enhancer, as well as in the PSE, which functions

like a TATA box (Hernandez, 2001). Competition experiments further showed

that the enhancers of the two promoters bind to common transcription factors

in vivo and in vitro (Carbon et al., 1987). These findings from the snRNA

promoters have led to the concept that common factors are shared by the Pol

II and III transcription machineries. The results of the current study using the

H1 promoter demonstrate that a RNA polymerase II enhancer is able to

enhance transcriptional activity of a RNA polymerase III promoter. An effective

knockdown of the luciferase reporter gene expression was demonstrated in

the current study, indicating the feasibility of using recombinant baculovirus

with engineered expression cassette as a delivery vector for RNA i. Since this

“proof of concept” study is based on the knockdown of a reporter gene, in

future, studies based on RNAi targeting aberrantly overexpressed genes, for

example, EGFR, VEGF, telomerase, or leptin (Kang et al., 2006; Saydam et al.,

2005; Tao et al., 2005; Pallini et al., 2006; Brown et al., 2005) would be


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necessary to further explore the feasibility of baculovirus-mediated RNAi for

gioma therapy.

Owing to their high transduction efficiency in vitro and in vivo, viral vectors

such as retrovirus and adenovirus are predominantly used in the glioma gene

therapy (Gomez-Manzano et al., 2004; Pulkkanen et al., 2005). However,

endogenous virus recombination, insertional mutagenesis, pre-existing

immune response and limited DNA-carrying capacity remain notorious

shortcomings of these viral vectors. The application of retrovirus vectors is

further hindered by the disadvantages such as low virus titer and instability of

the virion (Ram et al., 1997). For adenovirus, although several harmful

non-essential viral genes have been deleted, the strong immune response

caused by this virus still restricts its application, especially in human clinical

trials. Being able to overcome these practical limitations to some extent,

baculoviral vectors are attractive alternative vectors for gene therapy. A

tropism toward glial cells but not toward neurons has been demonstrated in

vivo and according to the results of the current study, this cell type specificity

could be enhanced by the use of a cell type-specific expression cassette. In

addition, most baculoviral vectors stay episomally in the nucleus and can thus

mediate only transient gene expression, which is suitable for cancer gene

therapy requiring short-term therapeutic gene expression. In terms of

transduction efficiency, baculoviral vectors tested in the current study are quite

comparable to other commonly used viral vectors, such as adeno-associated


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virus (AAV), adenoviruses, and lentivirus. As demonstrated in one recent study,

AAV2 was able to transduce up to 80% of cells at an MOI of 5,000, and 90% at

an MOI of 50,000 in five human glioma cell lines (Huszthy et al., 2005). In

another study (Enger et al., 2002), six glioma cell lines have been transduced

with AAV2 and adenovirus carrying GFP at an MOI of 100 and results showed

various transduction efficiencies in different cell lines, ranging from 1.5% to

81%, with most of the values being less than 50%. Transduction efficiency

from 50% to 90% in U87 glioma cells has been obtained with lentiviral vectors

at an MOI of 1 (Steffens et al., 2004). Furthermore, the large cloning capacity

of baculovirus makes it possible to incorporate several therapeutic genes

towards multiple therapeutic targets into a single baculoviral vector. Thus

baculoviral vectors provide a highly appealing possibility to improve the

therapeutic effects by using a combination of synergic therapeutic genes

based on different mechanisms. For example, we could incorporate an

expression cassette of the DTA gene and that of shRNA targeting EGFR into

one single baculoviral vector and thus inhibit the tumor growth synergistically.

In conclusion, a new type of recombinant baculoviral vector has been

developed by incorporating engineered regulatory elements. Its ability to

knockdown target gene expression was demonstrated both in vitro and in vivo;

hence it provides a potential therapeutic approach to knock down

disease-related genes. In addition, a baculoviral vector carrying a toxin gene

has been developed which could effectively inhibit glioma cell growth. To the


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best of our knowledge, this study is the first demonstration of the use of

baculovirus as a vector for cancer gene therapy in an animal model. It will

provide valuable knowledge for potential human clinical trials. Because of the

unsatisfactory outcomes of glioma gene therapy in clinical trials, it is necessary

to develop efficient and safe vectors that could be applied in the clinical setting.

In addition, the combination of suicide gene therapy with other approaches

such as immunotherapy, RNAi-based approaches, and/or conventional

treatments (e.g., surgery or chemotherapy) might be a more effective strategy

for glioma treatment.

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