Recombinant Baculovirus as a Highly Potent Vector for Gene Therapy of Human Colorectal Carcinoma: Molecular Cloning, Expression, and In Vitro Characterization

RESEARCH

Recombinant Baculovirus as a Highly Potent Vector for GeneTherapy of Human Colorectal Carcinoma: Molecular Cloning,Expression, and In Vitro Characterization

Arghya Paul • Barbara A. Jardin • Arun Kulamarva •

Meenakshi Malhotra • Cynthia B. Elias •

Satya Prakash

Published online: 9 February 2010

� Springer Science+Business Media, LLC 2010

Abstract Present therapeutic strategies for most cancers

are restricted mainly to the primary tumors and are also not

very effective in controlling metastatic states. Alternatively,

gene therapy can be a potential option for treating such

cancers. Currently mammalian viral-based cancer gene

therapy is the most popular approach, but the efficacy has

been shown to be quite low in clinical trials. In this study, for

the first time, the insect cell-specific baculovirus Autographa

californica multiple nucleopolyhedrovirus (AcMNPV) has

been evaluated as a vector for gene delivery to colorectal

cancer cells. Experiments involving factorial design were

employed to study the individual and combined effects of

different parameters such as multiplicity of infection (MOI),

viral incubation time and epigenetic factors on transduction

efficiency. The results demonstrate that baculovirus gene

delivery system holds immense potential for development of

a new generation of highly effective virotherapy for colo-

rectal, as well as other major carcinomas (breast, pancreas,

and brain), and offers significant benefits to traditional ani-

mal virus-based vectors with respect to safety concerns.

Keywords Colorectal cancer � Baculovirus � Gene

therapy � BacMam � Transduction � Biosafety �Therapeutic delivery

Introduction

Colorectal cancer is the third most commonly diagnosed

cancer and the third leading cause of cancer death in both

men and women in the United States, with about 148,810

new cases and 49,960 deaths expected in 2008 out of which

72% of cases arise in the colon and about 28% in the

rectum [1]. The most common practice to treat such can-

cers is surgical resection of the primary tumor. But this

often turns out to be inefficient because of the tumor’s

progress to metastases after attempted curative resections.

Although adjuvant chemotherapy or chemo-radiation had

shown some promises, still the long-term survival from

colon cancer has remained almost static [2]. Thus, new

measures are urgently required. Gene therapy represents a

logical approach to address such concerns, which may act

as an adjunct or conventional treatment [3–5].

Cancer gene therapy involves the inclusion of foreign

therapeutic genes into cancer cells resulting in temporary

suppression or complete eradication of the tumor. However,

the success of this therapy relies greatly on the efficiency and

selectivity at which therapeutic genes are introduced into the

tumor cells without causing any adverse side effects. Thus

efficient delivery and good biosafety profiles are two very

Arun Kulamarva and Meenakshi Malhotra contributed equally

to this work.

A. Paul � B. A. Jardin � A. Kulamarva � M. Malhotra �S. Prakash

Biomedical Technology and Cell Therapy Research Laboratory,

Department of Biomedical Engineering and Artificial Cells

and Organs Research Centre, Faculty of Medicine,

McGill University, 3775 University Street,

Montreal, QC H3A 2B4, Canada

C. B. Elias

Process Development, Sanofi Pasteur, Connaught Campus,

1755 Steeles Avenue West, Toronto, ON M2R 3T4,

Canada

C. B. Elias � S. Prakash (&)

Centre for Biorecognition and Biosensors, Biomedical

Engineering and Artificial Cells and Organs Research Centre,

Faculty of Medicine, McGill University, 3775 University Street,

Montreal, QC H3A 2B4, Canada

e-mail: [email protected]

Mol Biotechnol (2010) 45:129–139

DOI 10.1007/s12033-010-9248-7

important criteria during the selection of a delivery system.

Till date adenoviruses have been commonly used for a wide

variety of gene therapy applications; however, its transduc-

tion efficiency in solid tumors has been limited, resulting in

little evidence supporting significant clinical benefits [6–8].

Although researchers are trying to implement replication-

competent oncolytic viruses for cancer gene therapy [9–15],

its self-perpetuating nature in mammalian cells remains a

major concern for safety. Consequently, gene delivery into

tumors although conceptually elegant, has not yet achieved

its expected potential. Under such circumstances, targeting

tumor cells with insect-specific viruses, such as baculovi-

ruses, can become a potentially powerful tool. Since bac-

uloviruses are highly restricted insect viruses, which only

enter but not replicate in mammalian cells, therefore they

work best with priority to the efficacy and safety concerns of

cancer gene therapy. The natural promoters of these viruses

are not active in mammalian cells, as reported, there had been

no detection of expression from the polyhedrin promoter in

mammalian cells like Huh7 cells [16]. On the other hand, it

has shown to deliver genetic materials in vitro and in vivo,

into both dividing and nondividing mammalian cells, which

can be beneficial in case of prostate cancer that propagates at

a slower rate [17]. Earlier, recombinant baculoviruses have

shown to produce functional monoclonal antibody, by

infecting insect cells, which can recognize human colorectal

carcinoma cells [18]. Lyophilized preparation of insect cells

infected with baculovirus producing interferon-beta can

suppress the progressive growth of established orthotopic

tumors of colon cancer cells and occult liver metastasis in

vivo [19]. As known through literature, a combination

treatment of baculovirus gene therapy and chemotherapy

could trigger apoptosis in osteogenic sarcoma cells demon-

strating the potential of baculovirus as a cancer gene therapy

tool [20]. Recently, baculoviruses have also revealed its

potential to suppress gastric cancers in xenografted nude

mice [21].

Due to its growing popularity and potential scope in

cancer gene therapy, we have focused on exploring and

optimizing the combinatorial effects of different transduc-

tion factors using factorial design experiments in order to

enhance the therapeutic effects of baculovirus gene delivery

in treating cancers, particularly colorectal cancers. In this

study, we have investigated and compared the applicability

of baculoviruses as a potent gene therapy agent for some

unexplored, yet common cancers in detail. To perform these

studies, we have constructed recombinant baculoviruses

carrying Monster Green Fluorescent Protein as the reporter

transgenes, driven by the human cytomegalovirus (CMV)

immediate-early promoter. As this insect cell originated

baculovirus can express its transgene in mammalian cells,

driven by the CMV promoter, it is also known as BacMam

virus.

Materials and Methods

Cells and Media

Human colon cancer cells (SW480), breast cancer cells

(SkBr3), neuroblastoma cancer cells (Neuro2a), hepato-

cellular carcinoma cells (HepG2), and pancreatic carcinoma

cells line (PANC-1) were obtained from ATCC. SW480

was maintained in Leibovitz’s L-15 medium, SkBr3 was

maintained in McCoy’s 5A medium, Neuro2a in Eagle’s

Minimum Essential Medium (EMEM), HepG2 in Minimum

Essential Medium Alpha (MEM), and PANC-1 cells were

grown in Modified Eagle’s Medium (DMEM). All the

media were obtained from ATCC and supplemented with

10% Fetal Bovine Serum (FBS; Gibco, Invitrogen). The

cells were routinely maintained as stationary cultures in

75 cm2 cell culture flasks (Corning, NY), incubated at

37 �C in a controlled environment with 5% CO2, except in

case of SW480 where it did not require additional supply of

CO2. Spodoptera frugiperda (Sf9) insect cells (Invitrogen

Life Technologies, Carlsbad, CA) were maintained at 27 �C

in SF900-III SFM (Gibco, Life Technologies, Grand Island,

NY, USA) medium without FBS, using routine procedures

[22]. The Sf9 cells were maintained in exponential growth

phase and subcultured twice per week in 75 cm2 cell culture

flasks with 10 mL as working volume. Larger volumes were

prepared in 125 mL shaker flasks (Erlenmeyer, Corning) as

suspension culture, which were agitated at 115 rpm in a

horizontal incubator shaker with a working volume of

25 mL.

Cloning, Generation, and Quantification

of Recombinant MGFP-BacMam Baculoviruses

(Bac-MGFP)

The phMGFP vector carrying the Monster Green Fluores-

cent Protein (MGFP) and pVL1392 transfer vector, as

depicted in Fig. 1, were procured from Promega (Madison,

WI, USA) and BD Biosciences, respectively. Both the

phMGFP vector, harboring the PCMV promoter with hMGFP

gene, and the pVL1392 vector were digested with BglII and

XbaI restriction enzymes (New England Biolabs, Berverly,

MA). The excised PCMV-hMGFP gene and digested pVL1392

vector were purified and a basic ligation reaction with T4

DNA ligase (Promega) was performed to subclone the PCMV-

hMGFP gene into the pVL1392 transfer vector by directional

cloning method to generate the pVL1392-PCMV-PMGFP

transfer vector. The recombinant transfer vector was

amplified by transformation into the high efficiency DH5 acompetent E. coli (Invitrogen) by heat shock method. The

plasmids were then extracted and purified with QIAprep spin

miniprep kit (Qiagen Sciences, MD).

130 Mol Biotechnol (2010) 45:129–139

To generate the recombinant MGFP baculoviruses,

1 9 106 Sf9 cells were seeded in six well plates. A trans-

fection mixture comprised of 1 lg of the constructed

BacMam transfer vector, linearized baculovirus DNA (BD

Baculogold) and 6 lL of Cellfectin transfection reagent,

diluted in 200 lL media (Invitrogen Life Technologies,

Carlsbad, CA) was prepared. The mixture was incubated

for 45 min at room temperature and then was co-trans-

fected to the seeded cells. After 5 h of incubation at 27 �C,

the transfection media was replaced with Sf900 III media

and cells were further incubated at 27 �C for the recom-

bination event to occur inside the transfected insect cells

between the transfer vector and the linearized viral DNA.

This generates the newly formed active recombinant viru-

ses that start their replication process from within the insect

cells. Eventually the virus buds out of the cell and begins a

secondary round of infection in the nearby cells and so on.

96 h post-transfection (hpi), the cell cultures were col-

lected, centrifuged at 1000g for 10 min and then the

supernatant containing the baculovirus was filtered through

a 0.22-lm membrane and stored at 4 �C (P0 Virus Stock).

The P0 stock was further amplified from time to time by

using routine procedures as described previously [23].

Briefly, Sf9 cells were grown to mid-exponential phase and

diluted to 2 9 106 cells/mL with fresh SF900III medium.

The cultures were infected with P0 virus stock at a multi-

plicity of infection (MOI = plaque forming unit [pfu]/mL)

of 0.1. The supernatant containing the recombinant bacu-

lovirus was harvested 72 hpi by centrifugation. The viral

titre (pfu/mL) of the amplified viral stock was then deter-

mined using the Baculovirus Fast Plax Titer Kit (Novagen,

Madison, WI) according to the manufacturer’s protocol.

Bac-MGFP Transduction of Mammalian Cell Lines

Fluorescent expression levels of transduced cells were

quantified using plate reader Victor3 Multi Label Plate

Counter (Perkin Elmer, USA). Then, 2 9 104 cells were

seeded in each well in a 96 well plate and transduced with

100 lL PBS (no Ca and Mg) containing Bac-MGFP at

MOI ranging from 10 to 1000. After 8 h of incubation of

the cells with viruses at 25 �C, the cells were washed twice

with PBS and replenished with 200 lL of fresh medium

with or without 10 mM sodium butyrate (NaBu). The

relative fluorescence units of each well were measured

24 h post-transduction (hpt). This experiment was done in

triplicates, and the mean was calculated. Another set of

similar experiment was set up to find out the percentage of

cells transduced with Bac-MGFP, 24 hpt. In this set, the

monolayers were washed and the average percentage of

green fluorescent cells per high-power field were quantified

using fluorescence microscope (Nikon Eclipse TE2000-U);

five fields were counted per well and the mean value was

considered as the percentage transduced value.

To detect the fluorescence expression at the transcript

level, the cells were seeded in 6-well plates at 0.5 9

106 cells/well. Prior to transduction, the cell culture med-

ium was replaced by 500 lL of PBS containing Bac-MGFP

virus at varied MOI ranging from 100 to 1000. After 8 h of

viral incubation at 25 �C, the viral solution was removed

and the cells were washed twice with PBS and replenished

either with 2 mL fresh medium or with medium supple-

mented with 10 mM NaBu.

Application of Stat-Ease Design-Expert Software

for Optimization of Transduction Conditions

Optimization of the transduction procedure taking different

factors into account was done using the Design-Expert

software Version 7.1 from Stat-Ease. This software tool

allows the screening of multilevel factorial designs that

helps to look for the critical parameters which may indi-

vidually or synergistically affect a process [24]. In our

study, (i) MOI (ranging from 10 to 1000), (ii) NaBu con-

centration (ranging from 0 to 10 mM), and (iii) viral

incubation time (ranging from 2 to 8 h) were selected as

the three parameters affecting the transduction process

directly or indirectly. For this, SW480 cells were taken and

PVL1392(9639 bp)

phMGFP(4701 bp)

AmpR

PolyhedrinPromoter

MCS(BglII and XbaI)

CMV Promoter

hMGFP (684bp)

AmpR

BglII

XbaI A B

ColE ori

Fig. 1 Schematic

representation of starting

vectors and cloning strategy

for the PCMV-hMGFP/pVL1392

plasmid. The reporter gene

hMGFP was excised along with

the CMV promoter from

phMGFP Vector (a) using BglII

and XbaI restriction enzymes

and inserted in the Multiple

Cloning Site (MCS) of the

baculovirus transfer vector

pVL1392 (b)

Mol Biotechnol (2010) 45:129–139 131

transduced with baculovirus in 96 well plates. Based on

preliminary results, a two level factorial design with 20

experimental runs and four centre points was implemented.

The chosen was percentage of cells expressing GFP. The

statistical analysis of variance (ANOVA) for the responses

(transduction percentage) was generated by the above

mentioned software. Significant factors were established

where p \ 0.01.

Transcriptional Activity Analysis by RT-PCR

For detection of MGFP gene expression, total RNA was

extracted from transduced, mock-transduced and control

cells cultured in 6-well plates using RNA Extraction kit

(Qiagen) according to the manufacturer’s instructions. The

obtained RNA was then reverse transcribed to corresponding

cDNA using the Qiagen’s Reverse Transcription (RT) kit

following the supplied instructions. Further, PCR was per-

formed on the reverse transcribed product using Taq DNA

Polymerase (Invitrogen). To detect specific gene products,

the following forward (FP) and reverse primers (RP) were

used in PCR reactions. For Human Glyceraldehyde-

3-phosphate dehydrogenase (GAPDH) gene (112-bp prod-

uct), the FP was 50-TCAAGGGCATCCTGGGCTAC-30 and

RP was 50-AAGTGGTCGTTGAGGGCAATG-30; for the

MGFP gene (133-bp product) the FP was 50-GATGCAGC

GCAAGACCCTAAAG-30 and the RP was 50-GGTCTTG

AAGTCGCAGCGGTAG-30. Amplifications were carried

out for 25 cycles at 94 �C for 35 s (denaturation), 55 �C for

35 s (annealing), and 72 �C for 25 s (extension).

Results

Generation of Recombinant Baculoviruses

by Co-Transfection in Insect Cells

The pVL1392-PCMV-PMGFP recombinant transfer vector

was constructed by using the two vectors depicted in Fig. 1.

The confirmation of the correct insert in desired orientation

in the transfer vector was done by digesting the vector with

different sets of enzyme combinations and examining the

right band sizes through the agarose gel electrophoresis. The

purified pVL1392-PCMV-PMGFP transfer vector was then

co-transfected with linearized baculovirus DNA in the

insect cells to generate recombinant baculoviruses harboring

PCMV–MGFP genes. After 96 h, P0 viral stock was har-

vested from the culture collection of supernatant following

centrifugation. P0 viral stock was used to infect Sf9 cells in

a T-25 flask at MOI of 0.1 that resulted in a higher titre P1

viral stock, which was further amplified to achieve P2 viral

titre of 2.2 9 109pfu/mL as determined by the viral titration

kit.

Combinatorial Effects of Viral Dose, Viral Incubation

Time, and NaBu Concentration on Transduction

Efficiency: Factorial Matrix Design Experiments

The full factorial design was implemented to identify the

parameters having significant effects on increasing the

transduction efficiency. The factors considered were MOI,

viral incubation time and NaBu concentration at two levels

in a factorial design. The response factor studied was on

the transduction efficiency of SW480 colorectal cancer

cells. The three factors and their corresponding ranges are

mentioned in Table 1. Results under the condition of 8 h

viral incubation are shown in Fig. 2a. The two-dimensional

graph demonstrates that the MOI and NaBu concentration

both have significant positive effects on the transduction

percentage (response). Also their combined effect had a

greater impact in boosting the response compared to each

of them individually. This is clear from the graph (Fig. 2a)

where at MOI of around 750 and NaBu concentration of

5 mM, the transduction efficiency was around 70%,

whereas in absence of NaBu the efficiency dropped down

to approximately 50%. Graph of Fig. 2b depicts the pattern

of increment of the response level with increasing incu-

bation time and NaBu concentration under constant MOI of

1,000. The pattern suggests that increasing the incubation

time can have significant positive effect on the transduc-

tion, as it allows more viral particles to enter the host cell,

which in turn allows more expression of the transgene. The

transduction can be further boosted by addition of epige-

netic factor NaBu. This is evident from the graph, where

with a constant incubation time of 8 h, transduction effi-

ciency of around 73% can be increased to 85% in presence

of NaBu. Figure 2c explains that keeping the NaBu con-

centration constant, there can also be an increase in

transduction efficiency with increasing MOI and incubation

time. The two parameters are also dependant on each other

for maximum transduction. Like in the graph (Fig. 2c), a

low MOI (257.5) with less incubation time of 2 h gives us

low efficiency of approximately 37%, which can be

increased to around 49% with an increase in viral incuba-

tion time to 5 or 8 h. On the other hand, an increase in MOI

from 257.5 to 505 increases the transduction efficiency

from the range of 37–49% at constant incubation time of

Table 1 Factors used for the factorial design experiment and their

ranges

Factors Name Units Factors level range

Low Mean High

A MOI pfu/cell 10 505 1000

B NaBu mM 0 5 10

C Incubation time hour 2 5 8

132 Mol Biotechnol (2010) 45:129–139

5 h. Thus, Fig. 2(a–c) clearly illustrated that reducing the

values of one or more parameters in each case had sig-

nificant negative impact on the response value. It also

concluded that with an incubation time of 8 h, MOI 1000

and NaBu concentration of 10 mM, the transduction of the

cells reached its highest efficiency (*84%).

Transcriptional Activity Analysis of Baculovirus

Transduced Colorectal Cancer Cells

In order to do transcriptional analysis of the Bac-MGFP

transduced SW480 cells under different conditions reverse

transcriptase polymerase chain reaction (RT-PCR) was

Fig. 2 Optimization of

transduction efficiency of

SW480 colorectal cancer cells

using Bac-MGFP taking MOI,

viral incubation time and NaBu

as the three critically

influencing components.

(a, b, c) Two dimensional view

of numerical optimization

graphs show the combined

effect of two components on

transduction efficiency pattern

(measured in terms of %

Transduction) keeping the

third component constant

(8 h incubation time

in a; 1000 MOI in b;

10 mM NaBu in c)

each time. Here ANOVA

analysis software from Stat-

Ease Design Expert is used

Mol Biotechnol (2010) 45:129–139 133

performed. At 24 hpt, the transcription of MGFP gene was

done using RT-PCR. The 133 bp fragment was only found

in the sample of transduced SW480 cells indicating the

successful transcription of MGFP gene in cells (Fig. 3).

Further evaluation of the expression levels in SW480 cells

was determined by comparing the band intensities in

transduced cells, which revealed that with increase in

amount of MOI treatment, the amount of MGFP transcript

also increases. It also shows that addition of NaBu further

complements the increase in transcript levels. These data

were further reconfirmed by the fluorescence photographs

of the corresponding cells (Fig. 3a). The results were in

accordance with what we have obtained with protein level

expression as shown in Fig. 3a and b for colon cells.

Time Course Profile of Transgene Expression

in Transduced Colorectal Cancer Cells and Its

Dependence on Viral Incubation Time

In order to determine the duration of gene expression

mediated by Bac-MGFP, the SW480 cells were transduced

with an MOI of 1000 in a 96-well plate. Moreover, to find

out the dependence of gene expression on viral incubation

time the SW480 cells were transduced with Bac-MGFP at

varying incubation time (2, 5, and 8 h). The fluorescein

plate readings of each well were taken from time to time

(12 h, day 1, day 3, day 6, day 9, day 12, day 15, day 18,

day 21) using plate reader. The obtained average fluores-

cein values of each well were then normalized to that of the

wells having 8 h of viral incubation 24 hpt. The results as

represented in Fig. 4a shows a rapid high expression in the

cells on day 1, with higher incubation time giving the

higher expression (25% for 2 h; 57% for 5 h; 100% for

8 h). The expression gradually reduces with time and by

day 12 it almost ceases for the cells with viral incubation of

2 h. But with incubations of 5 and 8 h, the normalized

expression at day 12 still remained much higher to 10%

and 23% approximately. Thus the cells with 8 h incubation

time showed better normalized gene expression as well as

better persistence of the transgene with time, as studied in

21 days. These analyses were further reconfirmed by the

fluorescence photographs of the cells (Fig. 4b).

Fig. 3 a Transduction of Bac-MGFP on SW480 colorectal cancer

cells. The cells were transduced with Bac-MGFP for 8 h in PBS at

25 �C with and without NaBu treatment at an MOI of 50 and 100.

Transduced cells were photographed 24 hpt with fluorescence

microscope. Fluorescence images in the absence (top) and presence

(bottom) of phase contrast illumination are shown. b RT-PCR

analysis of MGFP gene expression 24 hpt with Bac-MGFP in Sw480.

The cells were given a viral incubation time of 8 h with different MOI

(50, 100, 500, and 1000).The cells treated with NaBu are compared

with nontreated cells. Controls include no template control for PCR

(NTC), no reverse transcriptase control for reverse transcription

reaction (-RT) and mock-infected cell control (Mock) for transduction

procedure. Amplification of housekeeping GAPDH gene serves an

internal control for the efficiency of reverse transcription reaction.

Molecular weight (Mw) markers are low range DNA ladder and the

sizes of the amplicon are indicated with arrows

134 Mol Biotechnol (2010) 45:129–139

Baculovirus-Mediated Superior Gene Transfer

to Colorectal Cancer Cells in Comparison to Breast,

Pancreas, and Neuronal Carcinomas

To explore the possibility of using baculovirus-mediated

gene delivery for a wide range of cancer cells, cell lines

from breast, pancreas, and neuronal tumor origins were

taken for study, apart from colorectal. The efficiency of

transduction, as evaluated by number of green fluorescent

cells, was determined 24 hpt and presented in Fig. 5

(in presence of NaBu) and Fig. 6 (in absence of NaBu).

The data show that the baculovirus vectors transduced

A

1

1

Nor

mal

ised

GF

P e

xpre

ssio

n (%

)

B

0

20

40

60

80

100

120

0

Day1 (5h)

3

8h

)

6 9Days po

5h

Day1 (8h)

9 12ost transductio

2h

D

15on

Day6 (8h)

18

Day

21

18 (8h)

Fig. 4 a Effect of viral

incubation time (in h) over time-

course profile of transgene

expression in SW480 cells. The

average expressed fluorescein

values were normalized to that

of cells with 1 day post

transduction, having 8 h of viral

incubation at 25 �C, and are

represented as normalized mean

expression in percentage value.

The cells were transduced with

Bac-MGFP for various

incubation periods. The data

represent the mean of three

independent culture

experiments ± Standard

Deviation (SD). b Transduced

cells with different incubation

times (denoted in brackets as 5

and 8 h) were photographed at

different time points(day 1, day

6, day 18) with fluorescence

microscope. Fluorescence

images in the absence (top) and

presence (bottom) of phase

contrast illumination are shown

Fig. 5 Quantification of

fluorescent cells (percentage

of total) with relation to MOI

(treated with 10 mM NaBu),

24 h post-transduction. SW480,

SkBr3, Neuro2a, and Panc1

cells were transduced with Bac-

MGFP at MOI of 100, 250, and

500 with viral incubation time

of 8 h at 25 �C. The cells were

then replenished with media

containing 10 mM NaBu. Data

represent the mean ± SD

Mol Biotechnol (2010) 45:129–139 135

SW480 cells with much higher efficiency than other cells.

At an MOI of 500, the baculovirus transduced 90 ± 2.5%

[standard deviation (SD), n = 3] and 37 ± 4.1% (SD,

n = 3) of the SW480 and Neuro2a cells, respectively, in

NaBu-treated cells. At MOI of 500, SkBr3 and Panc1 cells

show an efficiency of 40 ± 3.2% (SD, n = 3) and 31 ±

3.6% (SD, n = 3), respectively, in presence of NaBu.

SW480 cells showed almost equal transduction efficiency

with that of HepG2 cells, taken as a positive control

[25, 26]. The transduction efficiency appeared to have a

direct relationship with MOI in all the cell lines. To further

investigate, we checked the fluorescein expression of the

cells using plate reader 24 hpt as analyzed in Fig. 7. The

fluorescein expression level in SW480 cells treated with

10 mM NaBu was almost doubled as compared to NaBu

untreated cells. SW480 cells had three fold higher

expression levels in comparison to both Neuro2a and

SkBr3.

Discussions

In this report, we evaluated the application of baculovirus

gene therapy in diverse types of cancer (Table 2), where

the colorectal cancer cells showed highest transduction

efficiency in terms of GFP positive cells as well as its high

expression level as detected by fluorescein reading in the

plate reader. But leukemic cells like HL-60 (ATCC)

showed poor transduction with baculovirus vector (data not

shown). The study also focuses on investigating the critical

parameters that have individual as well as combinatorial

effects on the transduction level in the colorectal cancer

cells. The data show that the MOI, viral incubation time

and NaBu concentration have significant effects as mea-

sured by transduction efficiency and transgene expression

levels in the cells. We concluded that with an incubation

time of 8 h, MOI 1000 and NaBu concentration of 10 mM,

the transduction of the colorectal cancer cells reached its

highest efficiency. This data were also supported at the

transcript level as evaluated by RT-PCR in colon cancer

cells. The increase in viral incubation time also increased

the retention of transgene expression in transduced cells.

Similar observations were also noticed with other cancer

cells of different origins, which showed marked response in

transgene expression level and transduction efficiency with

varying viral dosage and treatment with NaBu.

The disparity in GFP expression among various cancer

cells was because each cell line has its unique ability to

Fig. 6 Quantification of

fluorescent cells (percentage

of total) with relation to MOI

(without treatment of 10 mM

NaBu), 24 hpt. SW480, SkBr3,

Neuro2a, and Panc1 cells were

transduced with Bac-MGFP at

MOI of 100, 250, and 500 with

viral incubation time of 8 h at

25 �C. The cells were then

replenished with media. Data

represent the mean ± SD

Fig. 7 Quantification of

fluorescent cells (fluoresccein

expression) with and without

10 mM NaBu treatment, 24 hpt.

SW480, SkBr3, Neuro2a, and

Panc1 cells were transduced

with Bac-MGFP at MOI of 500

with 8 h viral incubation time at

25 �C. The cells were then

replenished with media with or

without having NaBu in it. The

data represent the means ± SD

of triplicate samples

136 Mol Biotechnol (2010) 45:129–139

drive the CMV promoter to express the reporter gene.

Moreover, the uncoating efficiency of the baculovirus and

its genomic DNA movement into the host cell nucleus is

specific and unique to each cell-type. The colorectal cells

showed a rapid expression pattern with high-transduction

efficiencies which gradually diminished with time in a viral

incubation time dependant manner. The reason for this

transient expression could be attributed to the lack of nat-

ural mechanism of baculovirus to integrate with the host

genome, which is an inherent nature of most mammalian

viruses. Hence it does not exhibit long-term expression,

which arises out of genomic integration of the viral genome

into the host chromosome and its subsequent transfer to the

cell progeny. This is an important characteristic of the

insect cell-originating baculovirus with respect to its bio-

safety nature. Whereas most of the common mammalian

viral vectors have the probability to randomly integrate into

the host’s crucial genomic regions, thereby resulting in

unexpected tumerogenesis. Such transient expression can

be a limiting factor for cancer gene therapy, this problem

can be handled by using a hybrid baculovirus-adeno

associated viral (AAV) vector that harbors a gene cassette

flanked by AAV-inverted terminal repeats for prolonged

expression of transgene in host cells [27]. Moreover,

administration of multiple viral dosages can be another

solution to overcome the obstacle of transient expression.

Transduced cells exhibited marked increase in expres-

sion level with increase in viral dosage (MOI) and/or with

increase in viral exposure time. This was due to the

increase in MOI, wherein the virus to cell ratio increases

and thus in turn increases the chance of more number of

viral particles getting inside the cell and being transcribed.

Increasing the viral incubation time to more than 8 h did not

show any increase in transgene expression. This observa-

tion was understood as 8 h of incubation was optimal for

cellular uptake of viruses. Another reason could be that

extended viral exposure ([8 h) may pose a detrimental

effect on cells due to cytotoxicity. In order to detect the

false effects caused by down regulation of reporter gene

expression, induced by histone deacetylation condition of

the transduced cells expression, we performed studies at

both transcript and protein levels, in the presence and

absence of an epigenetic factor, NaBu. The most remark-

able effect of butyrate exhibited that epigenetic regulations

play an important role in influencing the transgene expres-

sion pattern. This chemical regulates histone deacetylation

by controlling the activation of histone deacetylase

enzymes [28]. It creates a histone hyperacetylation condi-

tion within the cells inducing a more relaxed chromatin

conformation conferring proper binding of transcription

factors to the promoter with consequential upregulation of

transgene expressions. That is why we noticed a marked

increase in transgene expression in all the transduced cell

lines in the presence of NaBu. Thus, we infer that epigenetic

phenomena, such as acetylation or compaction of chromatin

play an important role in baculovirus-mediated transgene

expression system. Proper chromatin state of the baculovi-

rus genome is thus crucial for efficient gene expression.

Despite its promising roles, baculovirus has still to over-

come some major obstacles, the lack of specific tumor tar-

geting being one of them. This has also been one of the

major reasons why the first generation nonreplicative ade-

noviruses showed low efficacy and disappointing clinical

trial data for cancer therapy [29]. Modifying the viral

envelope proteins to display tumor homing peptide ligands

on the baculovirus surface can be one way to increase

baculovirus transduction efficiency and circumvent the

problem [30].

There are several reports that have demonstrated the

broad range of cellular susceptibility of baculoviruses

toward human [31–34], porcine [35], rabbit [36], rodent

[37], avian [38], and fish [39] origins. This wide range of

permissive cell lines has encouraged researchers to extend

its applications toward cancer therapy. Current curative

strategies for most of the cancers are restricted to the pri-

mary tumor and early stages which are mainly treated by

surgical resection. As the effect of conventional treatments

like chemotherapy or radiotherapy to control metastatic

diseases are not sustained, therefore, gene therapy repre-

sents an alternative modality to treat cancer. Many safety

issues are under consideration with present mammalian

virus-based gene therapy trials [40]. On the contrary, insect

cell specific baculovirus plays an important role at this

juncture as a potential biologically safe gene delivery

Table 2 Baculovirus as a potential gene delivery vehicle for trans-

ducing different cancer cells

Origin Cell Line Gene therapy

Pancreas Panc-1

Neuronal Neuro2a

Colon/colorectal SW480

Breast SkBr3

Leukemic HL-60

Liver HepG2

Mol Biotechnol (2010) 45:129–139 137

vehicle. Fusing decay accelerating factor with baculovirus

gp64 gene under the control of polyhedrin promoter [41]

are shown to produce complement resistant baculoviruses.

This had actually resolved the major problem of in vivo

inactivation of baculovirus by the serum complements.

Cobra venom factor and anti-C5 antibody treatment as C

inhibitor [42] also shows to enhance transduction. Thus

baculovirus can emerge as an attractive gene delivery

system for cancer treatments [17, 43, 44].

Through this work, our data suggest for the first time

that baculovirus vectors could be used efficiently for

colorectal cancers. The future directions for this would be

the generation of recombinant baculovirus that can express

tumor suppressor genes like ATOH1 and APC for treating

colon cancers [45, 46], BRCA1 to treat breast cancers [47],

DPC4 for pancreatic cancer [48], p53 to treat hepatocar-

cinoma [49], and FKN to treat neuroblastoma [50]. Recent

concepts of targeting the colon cancer stem cells or using

suicide transgenes to induce antitumor effect are also some

of the promising alternative approaches where recombinant

baculovirus delivery system can be beneficial [27, 51].

Success of these studies will greatly improve the clinical

perspective of baculovirus-mediated gene therapy for can-

cer. Recombinant baculoviruses thus offers a potential

alternative to currently used adenoviral vectors for cancer

gene therapy. The increasing number of unique and specific

molecular targets for different cancer cells may prompt us

in future to design individualized baculoviruses for treating

respective cancer types with high efficacy. Such baculo-

viruses will not only make the delivery of genes restricted

and target specific, but will also increase the transduction

efficiency due to negligible loss to other nonspecific body

parts. This can further be boosted by the synergistic effect

of combination therapies comprising of radio-, chemo-, and

virotherapies.

Acknowledgments We gratefully acknowledge the assistance

received from the Natural Sciences and Engineering Research

Council of Canada (NSERC) to Dr. S. Prakash. A.P. acknowledges

financial support from Fonds de la recherche en sante du Quebec

(FRSQ), Quebec, Canada. A.K. acknowledges the Alexander Graham

Bell Post Graduate Scholarship—Doctoral from NSERC. M.M.

acknowledges the support of the McGill Faculty of Medicine Internal

Scholarship. The authors would like to thank B. Boulay, R. Tran, and

J. Montes from National Research Council Canada (NRC-BRI) for

their technical advice and assistance.

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