engleski

RETARGETING ADENOVIRUS TYPE 5 TO INTEGRIN α 4β 1

Gene therapy by means of recombinant adenovirus type 5 (Ad5) vectors could become a unique approach in treating certain types of cancer, among which acute myeloid leukemia (AML). To prove this statement valid, many experiments have been made in developing various recombinant Ad5 vectors and then applying them to cells in culture to see whether or not they would efficiently eliminate cancer cells. AML cells have increased expression of α 4β 1 (VLA-4) integrin on their cell surface for which targeting ligand CPLDIDFYC has been recently isolated. Therefore the aim of this research is to construct replication defective adenoviral vector by incorporating α 4β 1 integrin-targeting ligand into the HI-loop of the fiber protein, in order to obtain Ad5 vector that could efficiently infect AML cells. Construction of Ad5VLA4 was performed by inserting a duplex of oligonucleotides coding for CPLDIDFYC in portion of the Ad5 sequence encoding the HI loop and manipulation of the full length Ad5 genome as a stable plasmid in E. coli, using the bacterial homologous recombination machinery. All constructions were further confirmed by partial sequencing of the fiber gene. Both types of viruses, Ad5VLA4 and a control virus Ad5wt lacking targeting sequence, were multiplied in human embryonic kidney cell line-293 cell culture and purified by bending in CsCl gradients. The infectivity indices on 293 cells (the ratio of physical particles to infectious particles) of CsCl purified virus preparation were similar showing that incorporation of ligands in HI loop did not change virus infectivity. Ad5VLA4 will be further used for infection of human JURKAT cell line expressing VLA-4 to analyze whether the insertion of targeting ligand increased its transduction efficacy. If successful, we plan to construct another adenovirus vector that would express the herpes simplex thymidine kinase (HSV-TK) suicide gene that would make tumor cells sensitive to nucleoside analogs such as ganciclovir (GCV). This vector could be used in treating acute myeloid leukemia in vivo.

integrin α 4β 1 ; adenovirus ; retargeting

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