Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons

Recombinant adeno-associated viruses (rAA Vs) are promising vectors for gen e therapy since they efficiently and stably transduce a variety of tissues of immunocompetent animals. The major disadvantage of rAA Vs is their limit ed capacity to package foreign DNA (less than or equal to5 kb). Often, co-e xpression of two or more genes from a single viral vector is desirable to a chieve maximal Therapeutic efficacy or to track transduced cells in vivo by suitable reporter genes. The internal ribosome entry site (IRES) sequence of encephalomyocarditis virus has been widely used to construct bicistronic viral vectors. However, the IRES is rather long and IRES-mediated translat ion can be relatively inefficient when compared with cap-dependent translat ion. As an alternative to the IRES for in vivo gene expression, we studied the 16 amino-acid long 2A peptide of foot and mouth disease virus (FMDV). T he 2A peptide mediates the primary cis-'cleavage' of the FMDV polyprotein i n a cascade of processing events that ultimately generate the mature FMDV p roteins. We have generated several different rAA V genomes in which two cod ing regions are fused in-frame via the FMDV 2A sequence. We show that FMDV 2A efficiently mediates the generation of the expected cleavage products fr om the artificial fusion proteins in cells. Furthermore, we find that both EGFP and alpha -synuclein are expressed at substantially higher levels from 2A vectors than from the corresponding IRES-based vectors, while SOD-l is expressed at comparable or slightly higher levels. Finally, we demonstrate for the first time, that the 2A sequence results in effective bicistronic g ene expression in vivo after injection of 2A-dependent rAAVs into the rat s ubstantia nigra. We conclude that 2A-containing rAAVs may represent an attr active alternative to IRES-dependent vectors for ex vivo and in vivo gene e xpression and gene therapy.