Download Recombinant Baculovirus:

Surgical resection of primary tumor
 Chemotherapy

Inefficient- unable to control
metastasized tumors and promote longterm survival.
 Therefore, cancer is incurable using this
conventional methods.


Gene Therapy
› Either in combination with conventional
therapeutics
› Or as a replacement to these methods

The general goal of cancer gene
therapy is to introduce foreign
therapeutic genes into cancer cells in
order to temporarily suppress or
completely destroy the tumor.
Immunogene therapy
 Selective prodrug activation

› “suicide genes”
Tumor suppressor gene transfer
 Antisense techniques

› Inhibit activated oncogenes
Oncogene down- Vector-directed
regulation
cell lysis
2%
1%
Chemoprotection
5%
Tumor suppressor
gene
12%
Antisense
2%
Pro-drug
15%
Single chain
antibody
1%
Immunotherapy
62%
Safety
 Efficiency
 Specificity
 Expression level of transduced gene
 Simplicity of manufacture


Can be difficult to satisfy, depending on
therapeutic strategy and cancer type.
DNA/RNA viral and non-viral vectors.
 Mammalian viral-based cancer gene
therapy is increasingly popular.

› Fairly low efficiency in clinical trials
› Safety concerns

Another focus is on replicationcompetent oncolytic viruses.
› Perpetually exist in mammalian cells, safety
concerns.
Paul et al, primary paper.
 They evaluated the insect-cell specific
baculovirus as a vector for gene delivery
to colorectal cancer cells in addition to
other cancer types such as breast,
pancreas, and brain.

Baculovirus can be a powerful vector to
target tumor cells.
 It can enter mammalian cells without
replicating.

› Increased efficiency and eliminated safety
concerns with previous vectors
› Natural virus promoters are not active in
mammalian cells.

A recombinant baculovirus gene was
constructed with the Monster Green
Fluorescent Protein (MGFP) and the
cytomegalovirus (CMV) promoter.
CMV Promoter
MGFP Gene
phMGFP vector with the MGFP gene
and the CMV promoter
 pVL1392 baculovirus transfer vector
 Both digested with BgIII and Xball
restriction enzymes.
 The excised PCMV-hMGFP gene was
inserted into MCS of transfer vector.
 Amplification of recombinant transfer
vector in E. coli.

Now, the insect baculovirus is able to
express its transgenes in mammalian
cells, driven by the CMV promoter.
 The purified recombinant transfer vector
was co-transfected with linear
baculovirus DNA in Spodoptera
frugiperda (Sf9) insect cells to generate
recombinant baculoviruses with the
CMV promoter and hMGFP gene.


Human colon cancer cells (SW480),
breast cancer cells (SkBr3),
neuroblastoma cancer cells (Neuro2A),
hepatocellular carcinoma cells (HepG2),
and pancreatic carcinoma cells (PANC1) were maintained on appropriate
mediums with supplemented 10% Fetal
Bovine Serum (FBS) as stationary cultures.
Several different factors that may affect
transduction efficiency
 Multiplicity of Infection (MOI)- tested a
range of 10 -1,000
 Sodium Butyrate (NaBu) concentration in
medium- either 0 or 10 mM. NaBu has
various effects on cultured mammalian
cells, one of them is to increase gene
expression.
 Viral Incubation Time- 2-8 hours

SW480 cells were transduced with
baculovirus on 96-well plate.
 A plate reader was used to measure the
fluorescein readings from time to time to
determine the effect of viral incubation
time on gene expression.
 RNA was extracted from transduced cells
and Reverse Transcriptase Polymerase
Chain Reaction (RT-PCR) was performed
using MGFP specific primers for a 133 bp
product.

RT-PCR was also performed on control
samples, and the 133 bp MGFP fragment
was only found in the transduced SW480
cells. This is evidence that the MFGP
gene was successfully transcribed in the
cancer cells.
 By comparing band intensities from the
transduced cells, an increase in MOI and
addition of NaBu increased transcription
further.

This was confirmed by taking fluorescent
photographs of the cells with and
without phase contrast illumination.
 Higher MOI with addition of NaBu shows
the most fluorescence.

Higher MOI, NaBu, and longer
incubation time all had positive results on
transduction efficiency and maximum
transduction efficient occurred when
these parameters were combined
together.
 At an incubation time of 8 hours, the
highest efficiency achieved was with
MOI 1,000 and 10 mM NaBu.

Transduction efficiency much higher in SW480 cells

Although a high transduction efficiency
was observed, this gradually diminished
due to the fact that baculovirus does not
naturally integrate with the host
organism, so long-term expression was
not observed.

This is important in terms of safety
because the vector won’t randomly
integrate into crucial gene regions and
cause unexpected tumerogenesis,
whereas mammalian viral vectors would.
However, this is a limiting factor of
cancer gene therapy and can be
problematic.
 It can possibly be solved by using a
hybrid baculovirus-adeno associated
viral vector for more prolonged
expression.
 Also, multiple administrations of the viral
dose may also overcome transient
expression.


Baculovirus vectors can be used as safe
delivery vehicle for cancer gene
therapy, especially colorectal cancer
and possibly other cancer types in the
future.
The next step is to generate recombinant
baculoviruses that can express certain
tumor suppressor genes for specific
cancer types.
 Baculovirus may also be successful in
delivering suicide transgenes.




Paul, A.; Jardin, B. A.; Kulamarva, A.; Malhotra,
M.; Elias, C, B.; Prakash, S. Recombinant
Baculovirus as a Highly Potent Vector for
Gene Therapy of Human Colorectal
Carcinoma: Molecular Cloning, Expression,
and In Vitro Characterization. Mol
Biotechnol [Online] 2010, 45, 129 ff.
Zhang, J.; Russell, F. J. Vectors for Cancer Gene
Therapy. Cancer and Metastasis Reviews
[Online] 1996, 15, 385 ff.
Gunji, Y.; Ochiai, T.; Shimada, H., Matsubara, H.
Gene Therapy for Cancer. Surg Today
[Online] 2000, 30, 967 ff.