A NOVEL DICISTRONIC AAV VECTOR USING A SHORT IRES SEGMENT DERIVED FROM HEPATITIS-C VIRUS GENOME

Adeno-associated virus (AAV) vectors have a limited capacity for packa ging DNA. To insert both a therapeutic gene and a selectable marker ge ne in the same AAV vector efficiently, we developed a novel dicistroni c AAV vector containing a 230 base pairs (bp) internal ribosome entry site (IRES) element derived from hepatitis C virus (HCV) genome and a 420 bp blasticidin S-resistance gene (bsr) as a small selectable marke r in the second cistron. The 650 bp HCV IRES-bsr construct was placed downstream of the 3' end of the luciferase gene (Luc) under the contro l of the human cytomegalovirus (CMV) promoter. This dicistronic gene c onferred blasticidin S-resistance to 293 cells besides luciferase acti vity, when examined not only by transfection but also by transduction using AAV vectors. The dicistronic AAV vector harbouring HCV IRES-bsr is capable of expressing a therapeutic gene of up to similar to 3.6 ki lobases (kb) (including promoter/enhancer elements) as well as a selec table marker gene. If a selectable marker gene is not necessary, this vector is able to incorporate two different kinds of therapeutic genes more easily than that containing EMCV IRES. The dicistronic AAV vecto r described here is useful for expressing many kinds of cDNA besides a selectable marker. (C) 1997 Elsevier Science B.V.