Influenza A gutless vector: new approach against lung cancer

INTRODUCTION AND OBJECTIVES
Influenza A gutless vector: new approach against lung cancer
MATERIALS AND METHODS
•In vitro experiments
Francisco Latorre Pérez
Biotechnology Degree, Universitat Autònoma de Barcelona, 08193-
Bellaterra, Spain
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.
•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.
•Ex vivo experiments
Transfection into
packing cells
TK
PB2
PB1
HA PA
NP NS
M
NA
RNA polymerase I RNA polymerase II
(-) vRNA
with no
UTRs
TK (-) ssRNA
with HA3 UTRs
(packing signal)
Viral mRNA
TK mRNA
Viral proteins
+
TK protein
Budding
Transfection
into MDCK
MDCK (NBL-2)
•Suitable cell line as
transfection host.
•Normal phenotype
•Kidney cells from
Canis familiaris.
Infection with
therapeutic virus
NuLi-1 A549
Normal
phenotype
Malignant
phenotype
Thymidine kinase
production
Ganciclovir
killing ratio
GCV-P
GCV GCV GCV-PP GCV-PPP
TK
Nucleus
(division inhibition)
GCV-PP GCV-PPP Nucleus
(division inhibition)
GCV-P
GCV-P
Neighbor cell
(bystander effect)
•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.
MDCK (NBL-2) (ATCC® Number: CCL-34™)
NuLi-1 (ATCC® Number: CRL-4011™) A549 (ATCC® Number: CCL-185™)
vRNA
Genome
extraction
RT-PCR
x8 (-) ssRNA
x8 vDNA
Antisense
primer
Forward
primer
BsmBI
restriction site
BsmBI
restriction site
ORF
Amplification
Reverse
Transcription
5´
5´
5´
5´
5´
5´
5´
3´
3´
3´
3´
3´
3´
3´
x8 Amplicons
without 5’
and 3´ UTR
Digestion
with BsmBI
•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.
Construction of
suicide gene
Digestion with BspBI and BamH1 + Ligation
•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.
TK gene wt
Amplicon
for TK ORF
TK ORF BamHI
restriction site
BspHI
restriction site
5´
5´
5´
5´
5´
5´
3´
3´
3´
3´
3´
3´
Amplification
•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.
HA3 ORF BsmBI
restriction site
BsmBI
restriction site
BamHI
BspHI
5´ UTR 3´ UTR
Amplicon
for HA3
Ligation in
pHW2000
•Two types of
molecules are
synthetized
after ligation:
1. By RNA pol II: mRNA,
2. By RNA pol I: (-) ssRNA (only TK
plasmid has the UTRs necessary
to be packed).
Packing
RNA Polymerase I
RNA Polymerase II
Translation
Viral proteins
Nature Reviews Microbiology 6, 143-155 (February 2008)
* African Journal of Biotechnology Vol. 10 (24), pp. 5209-5218,13 June, 2011
pI tI
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).