Influenza A gutless vector: new approach against lung cancer

Influenza A gutless vector: new approach against lung cancer
Francisco Latorre Pérez
Biotechnology Degree, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain
INTRODUCTION AND OBJECTIVES
• Lung cancer is the most common cause of cancer-related death in men and women worldwide. The actual treatments for this disease are chemotherapy,
radiography and surgery; the main problem with them is that they are not always effective and can lead to bad secondary effects. Thus, there is an urgent
need in finding an effective and harmless alternative for lung cancer therapeutics.
• The aim of this project is to establish a different treatment against lung cancer, as a part of a final project report, based on one of the most promising new
therapies nowadays: virotherapy. Although the use of engineered virus has been reported in the consulted literature before, no satisfactory results have
been achieved. Thus, this work proposes the usage of a gutless engineered Influenza A/Aichi/2/68 H3N2, which has a natural tropism for lung, with the gene
coding for the thymidine kinase protein to infect a culture of human lung malignant cells and test the killing rate in presence of ganciclovir in media culture.
METHODOLOGY
• As a basis for the development of this work, many information sources such as paper reviews, patents, books, on-line official web pages, on-line catalogue
were consulted. Also, an interview with a specialist in virus-mediated gene therapy from the Institut Català d’Oncologia (ICO).
MATERIALS AND METHODS
• Ex vivo experiments
• In vitro experiments
TK
vRNA
•
•
5´
3´
5´
RNA polymerase I
3´
5´
3´
Antisense
primer
Amplification
Suitable cell line as
transfection host.
Normal phenotype
Kidney cells from
Canis familiaris.
RNA polymerase II
RT-PCR
3´
3´
NA
x8 (-) ssRNA
Reverse
Transcription
Forward
primer
M
PB2
Transfection
into MDCK
MDCK (NBL-2)
•
5´
NS
NP
PB1
MDCK (NBL-2) (ATCC® Number: CCL-34™)
Nature Reviews Microbiology 6, 143-155 (February 2008)
PA
HA
Transfection into
packing cells
Genome
extraction
(-) vRNA
with no
UTRs
x8 vDNA
5´
Viral mRNA
TK mRNA
TK (-) ssRNA
with HA3 UTRs
(packing signal)
5´
3´
3´
5´
BsmBI
restriction site
•
x8 Amplicons
without 5’
and 3´ UTR
Budding
BsmBI
restriction site
ORF
Viral proteins
+
TK protein
In this case, the 5´ and 3´UTRs are eliminated in each
amplicon due to the need to obtain viral genes
without the possibilities of being packed in the viral
capsids that they can form.
Digestion with BspBI and BamH1 + Ligation
Digestion
with BsmBI
A549
NuLi-1
Infection with
therapeutic virus
A549 (ATCC® Number: CCL-185™)
NuLi-1 (ATCC® Number: CRL-4011™)
Construction of
suicide gene
Ligation in
pHW2000
First, it is needed to do an amplification by
PCR of the thymidine kinase gene (TK);
adding two restriction sites for BspHI and
BamHI as shown below.
•
•
5´
3´
5´
3´
3´
5´
TK gene wt
3´
Amplification
5´
5´
3´
3´
5´
BspHI
restriction site
•
1.
2.
TK ORF
Amplicon
for TK ORF
RNA Polymerase II
tI
BsmBI
restriction site
HA3 ORF
Ganciclovir
killing ratio
Translation
3´ UTR
pI
•
RNA Polymerase I
•
Packing
GCV
GCV
Neighbor cell
(bystander effect)
Amplicon
for HA3
BsmBI
restriction site
Malignant
phenotype
TK
Viral proteins
BamHI
restriction site
BamHI
BspHI
Normal
phenotype
By RNA pol II: mRNA,
By RNA pol I: (-) ssRNA (only TK
plasmid has the UTRs necessary
to be packed).
Second, another RT-PCR for the virus
Hemagglutinin 3 (HA) gene is performed. In
this case, it is amplified the whole gene, with
5´ and 3´UTRs. The result is shown below.
5´ UTR
Thymidine kinase
production
Two types of
molecules are
synthetized
after ligation:
Nucleus
GCV-P
GCV-P
GCV-PP
GCV-PPP
(division inhibition)
GCV-P
GCV-PP
GCV-PPP
(division inhibition)
Nucleus
In order to see if the infection is efficient the killing ratio by ganciclovir (GCV) is tested.
Also, to test whether there is a differences between normal and malignant phenotypes,
two cell lines are used.
Level of formazan, product from tetrazolium salts reduction, in media culture is required
for this toxicity assay since it is only present in living, metabolically active cells.
* African Journal of Biotechnology Vol. 10 (24), pp. 5209-5218,13 June, 2011
DISCUSSION AND CONCLUSION
• Since there are no results, only conjectures can be made. Probably, gutless Influenza A vector with TK gene would be obtained, but the main problem of this
whole procedure is that the efficiency is very low, which means that in order to achieve a good level of therapeutic virus, high input is required. Then,
optimization is obligatory. In addition, counting the optimization time, this project was thought to be done in 3 years as a thesis project.
• In conclusion, despite the fact that some modifications of the process have to be done, this work shows a first attempt in the development of an Influenza
A gutless vector that hopefully could be used as a complementary treatment against lung cancer in the future.