Objective: Critical leg ischemia (CLI) represents the ultimate stage of peripheral arterial disease. Despite current surgery advances, patients with CLI have limited therapeutic options. Therapeutic angiogenesis thus appears as a powerful approach, aiming to stimulate vessel formation by angiogenic molecules administration. In this context, combined gene therapy has proved the most efficient. The present study aims to compare, in a preclinical mouse model, the therapeutic benefit of a combination of two angiogenic factors FGF2 (fibroblast growth factor 2) and Cyr61 using plasmid and viral vectors, able to generate short or long-term transgene expression in the leg, respectively.Methods: Two therapeutic genes, FGF2 and Cyr61, were introduced into IRES-based expression vectors (FGFiCyr) allowing co-expression of the two transgenes. The pro-angiogenic plasmid pC-FGFiCyr was assessed by intramuscular administration followed by electrotransfer into ischemic legs. To generate long-term transgene expression, the FGFiCyr bicistronic cassette was introduced into an adeno-associated virus-derived vector (rAAV). The rAAV treatment was performed either before, or immediately after, surgery. Therapeutic effects were analyzed by laser doppler imaging, clinical score and angiography.Results: The plasmid pC-FGFiCyr improved revascularization, reperfusion and clinical score. Surprisingly, when AAV-FGFiCyr was injected 21 or 28 days before surgery, the pro-angiogenic rAAV was drastically deleterious on all measured parameters. In contrast, when administrated shortly after surgery, AAV-FGFiCyr generated therapeutic benefits, with a significantly better clinical score than after treatment with the plasmid.Conclusions: Therapeutic effects of the angiogenic combination FGF2-Cyr61 is observed with short term transgene expression, but the treatment is significantly more efficient when a long-term expression viral vector is used. However, the rAAV FGFiCyr generated therapeutic benefit only when injected in an ischemic leg, whereas the same dose of rAAV exhibited deleterious effects when administrated to healthy animals. These data may contribute to the understanding of the moderate success of pro-angiogenic treatments in CLI gene therapy clinical assays.