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.