IRES-based Vector Coexpressing FGF2 and Cyr61 Provides Synergistic and Safe Therapeutics of Lower Limb Ischemia

Due to the lack of an adequate conventional therapy against lower limb ischemia, gene transfer for therapeutic angiogenesis is seen as an attractive alternative. However, the possibility of side effects, due to the expression of large amounts of angiogenic factors, justifies the design of devices that express synergistic molecules in low controlled doses. We have developed an internal ribosome entry site (IRES)–based bicistronic vector expressing two angiogenic molecules, fibroblast growth factor 2 (FGF2), and Cyr61. Through electrotransfer into the ApoE−/− mice hindlimb ischemic muscle model, we show that the IRES-based vector gives more stable expression than either monocistronic plasmid. Furthermore, laser Doppler analysis, arteriography, and immunochemistry clearly show that the bicistronic vector promotes a more abundant and functional revascularization than the monocistronic vectors, despite the fact that the bicistronic system produces 5–10 times less of each angiogenic molecule. Furthermore, although the monocistronic Cyr61 vector accelerates B16 melanoma growth in mice, the bicistronic vector is devoid of such side effects. Our results show an active cooperation of FGF2 and Cyr61 in therapeutic angiogenesis of hindlimb ischemia, and validate the use of IRES-based bicistronic vectors for the coexpression of controlled low doses of therapeutic molecules, providing perspectives for a safer gene therapy of lower limb ischemia

Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle

<p>Abstract</p> <p>Background</p> <p>Electrotransfer of plasmid DNA into skeletal muscle is a promising strategy for the delivery of therapeutic molecules targeting various muscular diseases, cancer and lower-limb ischemia. Internal Ribosome Entry Sites (IRESs) allow co-expression of proteins of interest from a single transcriptional unit. IRESs are RNA elements that have been found in viral RNAs as well as a variety of cellular mRNAs with long 5' untranslated regions. While the encephalomyocarditis virus (EMCV) IRES is often used in expression vectors, we have shown that the FGF-1 IRES is equally active to drive short term transgene expression in mouse muscle. To compare the ability of the FGF-1 IRES to drive long term expression against the EMCV and FGF-2 IRESs, we performed analyses of expression kinetics using bicistronic vectors that express the bioluminescent <it>renilla </it>and firefly luciferase reporter genes. Long term expression of bicistronic vectors was also compared to that of monocistronic vectors. Bioluminescence was quantified <it>ex vivo </it>using a luminometer and <it>in vivo </it>using a CCD camera that monitors luminescence within live animals.</p> <p>Results</p> <p>Our data demonstrate that the efficiency of the FGF-1 IRES is comparable to that of the EMCV IRES for long term expression of bicistronic transgenes in mouse muscle, whereas the FGF-2 IRES has a very poor activity. Interestingly, we show that despite the global decrease of vector expression over time, the ratio of firefly to <it>renilla </it>luciferase remains stable with bicistronic vectors containing the FGF-1 or FGF-2 IRES and is slightly affected with the EMCV IRES, whereas it is clearly unstable for mixed monocistronic vectors. In addition, long term expression more drastically decreases with monocistronic vectors, and is different for single or mixed vector injection.</p> <p>Conclusion</p> <p>These data validate the use of bicistronic vectors rather than mixed monocistronic vectors for long term expression, and support the use of the FGF-1 IRES. The use of a cellular IRES over one of viral origin is of particular interest in the goal of eliminating viral sequences from transgenic vectors. In addition, the FGF-1 IRES, compared to the EMCV IRES, has a more stable activity, is shorter in length and more flexible in terms of downstream cloning of second cistrons. Finally, the FGF-1 IRES is very attractive to develop multicistronic expression cassettes for gene transfer in mouse muscle.</p

Acute rimonabant treatment promotes protein synthesis in C2C12 myotubes through a CB1‐independent mechanism

International audienceSarcopenia is an age-related loss of muscle mass associated with changes in skeletal muscle protein homeostasis due to lipid accumulation and anabolic resistance; changes that are also commonly described in obesity. Activation of the endocannabinoid system is associated with the development of obesity and insulin resistance, and with the perturbed skeletal muscle development. Taken together this suggests that endocannabinoids could be regulators of skeletal muscle protein homeostasis. Here we report that rimonabant, an antagonist for the CB1 receptor, can prevent dexamethasone-induced C2C12 myotube atrophy without affecting the mRNA expression of atrogin-1/MAFbx (a marker of proteolysis), which suggests it is involved in the control of protein synthesis. Rimonabant alone stimulates protein synthesis in a time- and dose-dependent manner through mTOR- and intracellular calcium-dependent mechanisms. CB1 agonists are unable to modulate protein synthesis or prevent the effect of rimonabant. Using C2C12 cells stably expressing an shRNA directed against CB1, or HEK293 cells overexpressing HA-tagged CB1, we demonstrated that the effect of rimonabant is unaffected by CB1 expression level. In summary, rimonabant can stimulate protein synthesis in C2C12 myotubes through a CB1-independent mechanism. These results highlight the need to identify non-CB1 receptor(s) mediating the pro-anabolic effect of rimonabant as potential targets for the treatment of sarcopenia, and to design new side-effect-free molecules that consolidate the effect of rimonabant on skeletal muscle protein synthesis

Therapeutic potential of interfering with apelin signalling.

International audienceThe apelin receptor is a G protein-coupled receptor activated by several apelin fragments. Its tissue distribution suggests that apelin signalling is involved in a broad range of physiological functions. Endothelial cells, which express high levels of apelin receptors, respond to apelin through the phosphorylation of key intracellular effectors associated with cell proliferation and migration. In addition, apelin is a mitogen for endothelial cells and exhibits angiogenic properties in matrigel experiments. This review focuses on the therapeutic potential of apelin signalling, which is associated with pathologies that result from decreased vascularisation (ischemias) or neovascularisation (retinopathies and solid tumors)

Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle-2

<p><b>Copyright information:</b></p><p>Taken from "Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle"</p><p>http://www.biomedcentral.com/1472-6750/7/74</p><p>BMC Biotechnology 2007;7():74-74.</p><p>Published online 28 Oct 2007</p><p>PMCID:PMC2180170.</p><p></p> vectors (expression of LucF+ is under the translational control of EMCV, FGF-2 or FGF-1A IRES). Images are shown in pseudocolors. Time of exposure for each image and a pseudocolor scale are represented. *: non electrotransferred muscle. . Signal quantification was expressed in mean grey level/sec/ROI* (± sem). For each time point, n = 8 for EMCV, n = 10 for FGF-2 and n = 8 for FGF-1A. * Region Of Interest, corresponds to the bioluminescent area of the electrotransferred muscle

Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle-0

<p><b>Copyright information:</b></p><p>Taken from "Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle"</p><p>http://www.biomedcentral.com/1472-6750/7/74</p><p>BMC Biotechnology 2007;7():74-74.</p><p>Published online 28 Oct 2007</p><p>PMCID:PMC2180170.</p><p></p> promoter, encodes LucR in the first cistron (cap-dependent translation) and LucF (Fig. 1, 2) or LucF+ (Fig. 3, 4, 5) in the second cistron (IRES-dependent translation) [28, 36]. . muscle was taken from mice 5, 15 or 30 days after electrotransfer. LucR and LucF activities were measured from muscle extracts using a luminometer. LucR and LucF activities were expressed in Relative Luminescent Units per milligram of grinded muscle (RLU/mg) (see Mat. & Meth). Each value corresponds to an individual mouse from an experimental group (n = 5). . Mean values of the LucR (top) and LucF (bottom) activities have been represented for each experimental group presented in Fig. 1B (RLU per mg of muscle ± sem, n = 6). . It was obtained from the values shown in fig. 1B, using the following formula: LucF × 1000/LucR. For each time point, the median value was noted with a "-". No IRES activity could be calculated in the absence of detectable LucR activity

Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle-4

<p><b>Copyright information:</b></p><p>Taken from "Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle"</p><p>http://www.biomedcentral.com/1472-6750/7/74</p><p>BMC Biotechnology 2007;7():74-74.</p><p>Published online 28 Oct 2007</p><p>PMCID:PMC2180170.</p><p></p>cR or LucF+ (Mono LucR or Mono LucF, respectively), or with a mixture (30 μg + 30 μg) of the two plasmids (Mono LucR + LucF), or with 30 μg of the bicistronic plasmid containing the FGF-1 IRES (IRES FGF1A). LucR and LucF+ activities were measured from 5 to 21 days after electrotransfer. . LucF+ activity was detected at days 5, 15 and 21 in live animals as in Figure 3. Images are shown in pseudocolors. Time of exposure and a pseudocolor scale are represented. . On the top panel, the values detected with the CCD camera for each time point are expressed in mean grey level/second/ROI (mean ± sem, n = 6). The bottom panel shows the LucF+ relative quantification at day 5 (D5), day 15 (D15) and day 21 (D21) for each vector. . Two groups of mice were sacrificed at day 5 and 21, and muscles lysates were used for luciferase activity quantification using the luminometer. LucR (top panel) and LucF+ (bottom panel) are expressed in RLU/μg total proteins (mean ± sem, n = 6). . Luciferase relative quantification was obtained for each vector at day 5 (D5) and day 21 (D21)

Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle-3

<p><b>Copyright information:</b></p><p>Taken from "Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle"</p><p>http://www.biomedcentral.com/1472-6750/7/74</p><p>BMC Biotechnology 2007;7():74-74.</p><p>Published online 28 Oct 2007</p><p>PMCID:PMC2180170.</p><p></p>and LucF+ are expressed in RLU/μg total protein. Each value corresponds to one muscle. . It was determined as the ratio of LucF+/LucR multiplied by 100 (± sem, n = 6). Statistical anaysis, p = 0.0022 for EMCV, p = 1 for FGF1A and p = 0,0931 for FGF2

Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle-1

<p><b>Copyright information:</b></p><p>Taken from "Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle"</p><p>http://www.biomedcentral.com/1472-6750/7/74</p><p>BMC Biotechnology 2007;7():74-74.</p><p>Published online 28 Oct 2007</p><p>PMCID:PMC2180170.</p><p></p>tween the two activities. Linear regressions are shown with the regression coefficient R. The slopes of the regression straight lines are proportional to IRES activities at day 5