Use of the 2A sequence from foot-and-mouth disease virus in the generation of retroviral vectors for gene therapy

Abstract

We describe the construction of retroviral plasmid vectors lation gives rise to a bicistronic mRNA and two indein which two genes are linked by a minimum of 96 nucleopendent protein products. The system offers advantages tides encoding the 2A sequence from the picornavirus footto other alternative ways to create polycistronic mRNAs and-mouth disease virus (FMDV). Transcription and transand can be used in gene therapy delivery vectors.

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

“…However, this has not been observed for proteins and enzymes tested to date, including SOD-1 and EGFP (this study), chloramphenicol acetyltransferase, 37,39 38 puromycin Nacetyl transferase, ␤-glucuronidase and alkaline phosphatase. 40,41 Furthermore, our finding that genes can be expressed effectively when fused upstream or downstream of 2A suggests that modification-sensitive proteins could be encoded from the second cistron after 2A. This would result in the addition of only one proline to the N-terminus, which is not expected to interfere with protein function, in agreement with our findings and previous reports.…”

“…33,46 Regardless of the actual mechanism, the utility of the 2A sequence for the coordinate expression of two heterologous proteins has been demonstrated previously in plants 36,37 and cultured mammalian cells. [38][39][40] In this work, we have further explored the potential of the FMDV 2A sequence for the co-expression of two genes in cultured cells, and quantitatively compared the levels of second gene expression mediated by 2A-and IRES-dependent vectors for three different proteins. Furthermore, we generated 2A-dependent recombinant adeno-associated viruses and studied, for the first time, the utility of the 2A system for direct in vivo gene expression by intracerebral injection of rats with rAAV vectors carrying ␣-synuclein and EGFP coding sequences fused in-frame via 2A.…”

“…40 The small size of the 2A element may be useful for the coordinate expression of several enzymes to reconstitute a complete biosynthetic pathway, which may result in increased therapeutic benefit in some instances. 14,45 In tumor gene therapy, combinations of immunostimulatory, antiangiogenic and suicide genes may be expressed simultaneously from a single vector to maximize antitumor effects.…”

“…This would result in the addition of only one proline to the N-terminus, which is not expected to interfere with protein function, in agreement with our findings and previous reports. 37,38,40,41 The immunogenicity of the 2A peptide remains to be determined. A priori, we do not expect the 2A peptide to be more immunogenic than other foreign epitopes expressed as part of a transgene product.…”

“…These vectors were shown to co-express resistance and reporter genes in producer lines and infected cultured fibroblasts. 40,41 However, to date no results on the efficiency of 2A-mediated gene expression in animals and the potential of this system for in vivo gene therapy have been reported.…”