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See detailThe splicing factor TFIP11
Duchemin, Amandine ULiege

Speech/Talk (2019)

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See detailThe splicing factor TFIP11
Duchemin, Amandine ULiege

Poster (2019, February 01)

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See detailPhospho-Dependant Regulation Of TFIP11 Splicing Factor
Hanache, Sarah ULiege; Duchemin, Amandine ULiege; Peixoto, Paul et al

Poster (2019, February 01)

Pre-mRNA splicing is a fundamental process in mammalian gene expression contributing to protein diversity. Eukaryotic genes are alternatively spliced by the spliceosome, a large conserved machinery ... [more ▼]

Pre-mRNA splicing is a fundamental process in mammalian gene expression contributing to protein diversity. Eukaryotic genes are alternatively spliced by the spliceosome, a large conserved machinery complex comprising RNA, five small nuclear ribonucleoprotein particles (snRNPs: U1, U2, U4, U5 and U6) and around 200 proteins and splicing factors. These splicing factors are frequently phosphorylated by kinases. Such phosphorylation regulates their subcellular localization and interactions with target transcripts and protein partners, and thus regulates splicing reactions. Our lab demonstrated that the splicing factor TFIP11 controls cancer cell-cycle progression by regulating splicing of a specific subset of pre-mRNA (see A.Duchemin abstract). We and others have demonstrated that TFIP11 is highly phosphorylated. This phosphorylation might be critical for its nuclear localization, transport and/or its interaction with RNA or proteins such as DHX15. So, the present project is divided into three main goals: 1) Identification of kinases regulating phosphorylation of TFIP11 2) Identification of phospho-residues of TFIP11 and 3) Impact of phosphorylation on TFIP11 activity. Phosphoproteomic analyses have shown that at least 4 serine residues (S59, S96, S98, S210) and 2 tyrosine residues (Y162, Y722) on TFIP11 undergo phosphorylation. Interestingly, some of these potential phospho-residues are within functional TFIP11 domains including G-Patch domain, nuclear localization signal (NLS) and nuclear speckle targeting sites (NSTS) and belong to consensus sites for CK2 and Prp4K, two kinases known to regulate the activity of splicing factors. Immunofluorescence experiments demonstrated that CK2 and Prp4K colocalize with TFIP11 in the nuclear speckles. Co-immunoprecipitation experiments and/or proximity ligation assay (PLA) demonstrate that TFIP11, CK2 and Prp4K were associated in the same complex. Interestingly, we identified EFTUD2 as an interacting partner of TFIP11 and it seems that EFTUD2 coordinates the same splicing program as TFIP11. Finally, preliminary PLA data show that CK2 inhibition increases the interaction between TFIP11 and EFTUD2, suggesting that a CK2-dependent phosphorylation of TFP11 might be important to regulate its interaction with partner protein EFTUD2. Further investigations are ongoing to determine the functional role of the TFIP11-EFTUD2-CK2 complex in the regulation of the splicing program. [less ▲]

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See detailUnveiling of a new role for DPF3 protein in cancer biology
Verrillo, Giulia ULiege; Hanache, Sarah ULiege; Duchemin, Amandine ULiege et al

Poster (2019, February)

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See detailCationic Liposomes Carrying siRNA: Impact of Lipid Composition on Physicochemical Properties, Cytotoxicity and Endosomal Escape
Lechanteur, Anna ULiege; Sanna, Vincent; Duchemin, Amandine ULiege et al

Conference (2018, August)

In recent year, cationic liposomes have gained a lot of attention for siRNA delivery using different local routes of administration as the vaginal [1] or the pulmonary routes. However, lipoplexes have to ... [more ▼]

In recent year, cationic liposomes have gained a lot of attention for siRNA delivery using different local routes of administration as the vaginal [1] or the pulmonary routes. However, lipoplexes have to face several biological and intracellular barriers before releasing the genetic cargo. In this study, we focus our effort on intracellular barriers and more specifically on endosomal escape and cytosolic delivery of siRNA as well as on the cytotoxicity due to cationic lipids. Indeed, we have previously demonstrated that the surface charge of liposomes composed of the cationic lipid DOTAP is correlated to the induction of cytotoxicity [2]. In the present study, we have investigated the impact of different cationic lipids and co-lipids on the cytotoxicity and also on the endosomal escape of siRNA by flow cytometry, qRT-PCR and Western Blot assays [3]. To address these issues, we developed four liposomal formulations composed of two different cationic lipids (DOTAP and DC-Cholesterol) and different ratio of co-lipids (cholesterol and DOPE). Formulations were DOTAP/Cholesterol/DOPE 1/0.75/0.5, DOTAP/Cholesterol/DOPE 1/0.5/0.5, DOTAP/DOPE 1/1 and DC-Cholesterol/DOPE 1/1. Each type of liposomes were complexed to siRNA at six different N/P molar ratios and physico-chemical properties were characterized in terms of Z-average size, Zeta potential and complexation efficiency by gel retardation assay. Consequently, three N/P ratios (2.5, 5 and 10) were selected for in vitro experiments on A549 cells. First, we studied the cell viability of A549 cells treated during 24 h with liposomes complexed to inactive siRNA at different N/P molar ratios at siRNA concentrations of 40 and 100 nM. We have shown that the cytotoxicity is influenced by the N/P ratio, the concentration of cationic lipid as well as the nature of the cationic lipid. Secondly, the cellular uptake were evaluated by flow cytometry using the dry Trypan Blue® to quench the external fluorescence. Despite the fact the transfection rate were not significantly different, the mRNA knock-down efficiency were not similar between formulations. Liposomes containing 50% of DOPE induced a mRNA silencing of around 80% as well as the protein knock-down. This study allowed to highlight crucial parameters in order to develop lipoplexes which are safe and induce an efficient intracytoplasmic release of siRNA. Acknowledgments: The authors thank the Belgium National Fund for Scientific Research (FNRS, http://www.frs-fnrs.be) –Télévie for financial support and the Giga Cell Imaging and Flow Cytometry Platform for their collaboration. Anna Lechanteur is a FNRS-Télévie post-doctoral researcher. Amandine Duchemin is a FRIA FNRS Fellow. Denis Mottet is a FNRS Research Associate. References: 1. Lechanteur, A., Furst, T. Delvenne, P. et al., Promoting vaginal distribution of E7 and MCL-1 siRNA-silencing nanoparticles for cervical cancer treatment Molecular Pharmaceutics, 2017. 14: p. 1706-1717. 2. Lechanteur, A., Furst, T. Evrard, B. et al., PEGylation of lipoplexes: The right balance between cytotoxicity and siRNA effectiveness. Eur J Pharm Sci, 2016. 93: p. 493-503. 3. Lechanteur, A., Sanna, V. Duchemin, A. et al., Cationic Liposomes Carrying siRNA: Impact of Lipid Composition on Physicochemical Properties, Cytotoxicity and Endosomal Escape. Nanomaterials (Basel), 2018. 8(270): p. 1-12. [less ▲]

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See detailCationic Liposomes Carrying siRNA: Impact of Lipid Composition on Physicochemical Properties, Cytotoxicity and Endosomal Escape
Lechanteur, Anna ULiege; Sanna, Vincent; Duchemin, Amandine ULiege et al

in Nanomaterials (2018), 8(5),

Abstract: In recent year, cationic liposomes have gained a lot of attention for siRNA delivery. Despite this, intracellular barriers as endosomal escape and cytosolic delivery of siRNA still represent a ... [more ▼]

Abstract: In recent year, cationic liposomes have gained a lot of attention for siRNA delivery. Despite this, intracellular barriers as endosomal escape and cytosolic delivery of siRNA still represent a challeng, as well as the cytotoxicity due to cationic lipids. To address these issues, we developed four liposomal formulations, composed of two different cationic lipids (DOTAP and DC-Cholesterol) and different ratio of co-lipids (cholesterol and DOPE). The objective is to dissect these impacts on siRNA efficacy and cytotoxicity. Liposomes were complexed to siRNA at six different N/P molar ratios, physico-chemical properties were characterized, and consequently, N/P 2.5, 5 and 10 were selected for in vitro experiments. We have shown that cytotoxicity is influenced by the N/P ratio, the concentration of cationic lipid, as well as the nature of the cationic lipid. For instance, cell viability decreased by 70% with liposomes composed of DOTAP/Cholesterol/DOPE 1/0.75/0.5 at a N/P ratio 10, whereas the same formulation at a N/P ratio of 2.5 was safe. Interestingly, we have observed differences in terms of mRNA knock-down efficiency, whereas the transfection rate was quite similar for each formulation. Liposomes containing 50% of DOPE induced a mRNA silencing of around 80%. This study allowed us to highlight crucial parameters in order to develop lipoplexes which are safe, and which induce an efficient intracytoplasmic release of siRNA. [less ▲]

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See detailCationic Liposomes Carrying siRNA: Impact of Lipid Composition on Physicochemical Properties, Cytotoxicity and Endosomal Escape
Lechanteur, Anna ULiege; Sanna, Vincent; Duchemin, Amandine ULiege et al

in Cationic liposomes carrying siRNA: impact of lipid composition on physicochemical properties, cytotoxicity and endosomal escape (2018, April)

In recent year, cationic liposomes have gained attention for siRNA delivery using different local routes of administration as the vaginal [Lechanteur et al, Mol Pharm.2017] or the pulmonary routes ... [more ▼]

In recent year, cationic liposomes have gained attention for siRNA delivery using different local routes of administration as the vaginal [Lechanteur et al, Mol Pharm.2017] or the pulmonary routes. However, lipoplexes have to face several biological and intracellular barriers before releasing the genetic cargo. In this study, we focus our effort on intracellular barriers and more specifically on endosomal escape and cytosolic delivery of siRNA as well as on the cytotoxicity due to cationic lipids. Indeed, we have previously demonstrated that the surface charge of liposomes composed of the cationic lipid DOTAP is correlated to the induction of cytotoxicity [Lechanteur et al. EJPS.2016]. In the present study, we have investigated the impact of different cationic lipids and co-lipids on the cytotoxicity and also on the endosomal escape of siRNA by flow cytometry, qRT-PCR and Western Blot assays [Lechanteur et al. Nanomat.2018]. To address these issues, we developed four liposomal formulations composed of two different cationic lipids (DOTAP and DC-Cholesterol) and different ratio of co-lipids (cholesterol and DOPE). Formulations were DOTAP/Cholesterol/DOPE 1/0.75/0.5, DOTAP/Cholesterol/DOPE 1/0.5/0.5, DOTAP/DOPE 1/1 and DC-Cholesterol/DOPE 1/1. Each type of liposomes were complexed to siRNA at six different N/P molar ratios and physico-chemical properties were characterized in terms of Z-average size, Zeta potential and complexation efficiency. Consequently, three N/P ratios (2.5, 5 and 10) were selected for in vitro experiments on A549 cells. We studied the cell viability of cells with liposomes complexed to inactive siRNA at different N/P molar ratios at two siRNA concentrations. We have shown that the cytotoxicity is influenced by the N/P ratio, the concentration of cationic lipid as well as the nature of the cationic lipid. The cellular uptake were evaluated using the dye Trypan Blue® to quench the external fluorescence. Despite the fact the transfection rate were not significantly different, the mRNA knock-down efficiency were not similar between formulations. Liposomes containing 50% of DOPE induced a mRNA silencing of around 80% as well as the protein knock-down. This study allowed to highlight crucial parameters in order to develop lipoplexes which are safe and induce an efficient intracytoplasmic release of siRNA. [less ▲]

Detailed reference viewed: 34 (1 ULiège)
See detailUnveiling a New Role of DPF3 Protein in Cancer Biology
Verrillo, Giulia ULiege; Hanache, Sarah; Duchemin, Amandine ULiege et al

Poster (2018, February 01)

Detailed reference viewed: 40 (5 ULiège)
See detailDepletion of splicing factor TFIP11.
Duchemin, Amandine ULiege

Poster (2017, September 22)

Detailed reference viewed: 24 (9 ULiège)
See detailUnveiling a New Role of DPF3 Protein in Cancer Biology
Verrillo, Giulia ULiege; Hanache, Sarah; Duchemin, Amandine ULiege et al

Poster (2017, May 11)

Detailed reference viewed: 19 (4 ULiège)
See detailNew role for TFIP11 in
Duchemin, Amandine ULiege

Speech/Talk (2017)

Detailed reference viewed: 16 (5 ULiège)
See detailUnveiling a New Role of DPF3 Protein in Cancer Biology
Verrillo, Giulia ULiege; Duchemin, Amandine ULiege; Hanache, Sarah et al

Poster (2017, February 01)

Detailed reference viewed: 16 (3 ULiège)