ORIGINAL ARTICLE Efficient gene transfer in skeletal muscle with AAV- derived bicistronic vector using the FGF-1 IRES A Delluc-Clavie`res 1,2 , C Le Bec 3,6 , L Van den Berghe 1,2,6 , C Conte 1,2 , V Allo 3 , O Danos 4,5 and A-C Prats 1,2 1 Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), U858, Toulouse, France; 2 Team ‘Translational control and gene therapy of vascular and tumoral diseases,’ Institut de Me´decine Mole´culaire de Rangueil, IFR31, Universite´ Toulouse III Paul Sabatier, Toulouse, France; 3 Ge´ne´thon, Evry, France; 4 Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), U781, Paris, France and 5 Universite´ Paris 5, Paris, France IRESs (internal ribosome entry sites) are RNA elements behaving as translational enhancers in conditions of global translation blockade. IRESs are also useful in biotechnolo- gical applications as they allow expression of several genes from a single mRNA. Up to now, most IRES-containing vectors use the IRES from encephalomyocarditis virus (EMCV), highly active in transiently transfected cells but long and not flexible in its positioning relative to the gene of interest. In contrast, several IRESs identified in cellular mRNAs are short and flexible and may therefore be advantageous in gene transfer vectors such as those derived from the adeno-associated virus (AAV), where the size of the transgene expression cassette is limited. Here, we have tested bicistronic AAV-derived vectors expressing two luciferase genes separated by the EMCV- or fibroblast growth factor 1 (FGF-1) IRES. We demonstrate that the AAV vector with the FGF-1 IRES, when administrated into the mouse muscle, leads to efficient expression of both transgenes with a stable stoechiometry, for at least 120 days. Interestingly, the bicistronic mRNA containing the FGF-1 IRES leads to transgene expression 10 times superior to that observed with EMCV, in vivo. AAV vectors featuring the FGF-1 IRES may thus be advantageous for gene therapy approaches in skeletal muscle involving coexpression of genes of interest. Gene Therapy (2008) 15, 1090–1098; doi:10.1038/gt.2008.49; published online 27 March 2008 Keywords: gene transfer; AAV; bicistronic; IRES; muscle; FGF Introduction Optimization and safety of gene transfer is presently a challenge in gene therapy approaches. In particular, it can be useful to coexpress two or more genes of interest, in the case of heterodimeric proteins such as interleukin 12, when a resistance gene must be coexpressed, or to combine therapeutic molecules with a synergistic effect as reported for antitumoral antiangiogenesis. 1,2 Gene coexpression is also useful in the case of monogenic inherited diseases: coexpression of insulin-like growth factor 1 or antimyostatin with microdystrophin or calpain 3, respectively, appears as an attractive strategy to improve the gene therapy of myopathies. 3,4 Among the possibilities to obtain such a coexpression, one can use either two separate vectors or a single vector expressing two transcription units. However, these approaches can lead to transgene silencing, especially if the gene product shows toxicity. 5 Another approach is based on the use of internal ribosome entry sites (IRES), structural RNA elements that allow the translation machinery to be recruited within the mRNA, while the dominant pathway of translation initiation recruits ribosomes on the mRNA capped 5 0 end. 6,7 IRESs can therefore be used to design expression cassettes coding for several genes from a single transcription unit. 5,8 Such genes encoded by the same mRNA are called ‘cistrons’ and the corresponding mRNA is ‘bi- or multicistronic.’ The first IRESs were discovered in 1988 in picorna- viruses 9 and currently, most available bicistronic vectors contain the IRES from the encephalomyocarditis virus (EMCV). 10 This IRES is indeed very efficient in transi- ently transfected cells and has a higher activity than the poliovirus IRES in human embryonic stem cells. 11 Retro- viral IRESs have also been successfully utilized in the construction of high-titer retroviral vectors. 12 In addition, several IRESs with distinct features and properties have been described in cellular mRNAs. 7,13 Several of them are shorter than the EMCV IRES and more flexible with regards to the distance between the IRES and the initiation codon. 14 These cellular IRESs have often been reported as having a lower activity than the EMCV IRES in cell transfection assays. Yet these comparisons do not necessarily reflect physiological situations and we have previously shown that in transgenic mice cellular IRESs can display tissue-specific activities, in contrast to the EMCV IRES. 15 In addition, measurements of IRES activities after plasmid electrotransfer into mouse skele- tal muscle have revealed that the IRES from the fibroblast Received 14 August 2007; revised 26 January 2008; accepted 15 February 2008; published online 27 March 2008 Correspondence: Dr A-C Prats, Team ‘Translational control and gene therapy of vascular and tumoral diseases,’ Institut de Me´decine Mole´culaire de Rangueil, Universite´ Toulouse III Paul Sabatier, Inserm U858, IFR31, Avenue Jean Poulhes, BP 84225, Toulouse cedex 04 31432, France. E-mail: Anne-Catherine.Prats@toulouse.inserm.fr 6 These authors contributed equally to this work. Gene Therapy (2008) 15, 1090–1098 & 2008 Macmillan Publishers Limited All rights reserved 0969-7128/08 $30.00 www.nature.com/gt