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Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth.

Crombez L, Morris MC, Dufort S, Aldrian-Herrada G, Nguyen Q, Mc Master G, Coll JL, Heitz F, Divita G - Nucleic Acids Res. (2009)

Bottom Line: In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection.We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised.The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherches de Biochimie Macromoléculaire, Department of Molecular Biophysics and Therapeutic, UMR-5237 CNRS-UM2-UM1, 1919 Route de Mende, 34293 Montpellier, France.

ABSTRACT
The development of short interfering RNA (siRNA), has provided great hope for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake and bioavailability of siRNA remain a major obstacle to their clinical development and most strategies that propose to improve siRNA delivery remain limited for in vivo applications. In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection. Moreover, we show that functionalization of this carrier with cholesterol significantly improves tissue distribution and stability of siRNA in vivo, thereby enhancing the efficiency of this technology for systemic administration following intravenous injection without triggering any non-specific inflammatory response. We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised. The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.

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MPG-8-mediated delivery of siRNA targeting cyclin B1 induces G2-arrest. MPG-8 (A and B) and MPG (C) dose–response of Cyclin B1 silencing at the protein and mRNA levels. Stock solutions of MPG-8/siRNA (100 nM) or MPG/siRNA (500 nM) particles were prepared at a molar ratio of 1/20, and lower concentrations (from 200 nM to 0.125 nM) were obtained by serial dilution of the stock solution in PBS. HeLa (A) and HS-68 (B and C) cells (60% confluency) were overlaid with preformed complexes for 30 min, then fresh DMEM supplemented with 10% FCS was added directly to the cells, which were then returned to the incubator for 24 h. Cyclin B1 protein levels were determined by western blotting using Cdk2 as a control for quantification (grey bars). Cyclin B1 mRNA levels were measured 12 h after transfection using Quantigen technology (white bars). Mismatched Cyc-B3 siRNA associated with MPG-8 (200 nM) and empty MPG-8 particles (20 µM) were used as a control. Dose–response of G2-arrest associated with Cyclin B1 silencing (D). HeLa (grey bars) and HS68 (white bars) cells were treated with increasing concentrations of MPG-8/siRNA-Cyc-B1 from 0.25 to 20 nM. The cell cycle status was evaluated by FACS analysis. Mismatched Cyc-B3 siRNA (100 nM) and GAPDH siRNA (100 nM) associated to MPG-8 as well as to MPG-8 carrier alone (20 µM) were used as controls. Results are the means ± of four separate experiments.
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Figure 2: MPG-8-mediated delivery of siRNA targeting cyclin B1 induces G2-arrest. MPG-8 (A and B) and MPG (C) dose–response of Cyclin B1 silencing at the protein and mRNA levels. Stock solutions of MPG-8/siRNA (100 nM) or MPG/siRNA (500 nM) particles were prepared at a molar ratio of 1/20, and lower concentrations (from 200 nM to 0.125 nM) were obtained by serial dilution of the stock solution in PBS. HeLa (A) and HS-68 (B and C) cells (60% confluency) were overlaid with preformed complexes for 30 min, then fresh DMEM supplemented with 10% FCS was added directly to the cells, which were then returned to the incubator for 24 h. Cyclin B1 protein levels were determined by western blotting using Cdk2 as a control for quantification (grey bars). Cyclin B1 mRNA levels were measured 12 h after transfection using Quantigen technology (white bars). Mismatched Cyc-B3 siRNA associated with MPG-8 (200 nM) and empty MPG-8 particles (20 µM) were used as a control. Dose–response of G2-arrest associated with Cyclin B1 silencing (D). HeLa (grey bars) and HS68 (white bars) cells were treated with increasing concentrations of MPG-8/siRNA-Cyc-B1 from 0.25 to 20 nM. The cell cycle status was evaluated by FACS analysis. Mismatched Cyc-B3 siRNA (100 nM) and GAPDH siRNA (100 nM) associated to MPG-8 as well as to MPG-8 carrier alone (20 µM) were used as controls. Results are the means ± of four separate experiments.

Mentions: Dose–response experiments performed on cultured cells revealed that MPG-8-mediated delivery of siRNA (Cyc-B1) induced a robust biological response associated with downregulation of both cyclin B1 protein and mRNA levels (Figure 2A and B). An siRNA concentration of 5 nM was sufficient to reduce cyclin B1 levels by more than 85% in HeLa cells (Figure 2A) and IC50 of 1.1 ± 0.3 nM and 0.9 ± 0.2 nM were estimated for downregulation of protein levels, and of 0.6 ± 0.1 nM and 0.4 ± 0.1 nM for mRNA levels, for non-transformed HS68 fibroblasts (Figure 2B) and HeLa cells (data not shown), respectively. In comparison, when siRNA were delivered with MPGΔNLS, IC50 values of 24 ± 5 nM and 35 ± 7 nM were obtained for downregulation of protein and mRNA levels, respectively (Figure 2C). That MPG is 30- to 60-fold less efficient than MPG-8 can be directly correlated to differences in stability, solubility and size of the siRNA/MPG complexes. Reduction of cyclin B1 protein levels was directly associated with accumulation of cells with a 4N content, consistent with downregulation of Cdk1-Cyclin B1 activity, and was optimally obtained with 2 nM siRNA and IC50 values estimated to 0.8 ± 0.2 nM and 1.2 ± 0.4 nM for HeLa and HS68 cells, respectively (Figure 2D). In contrast, no effect on cyclin B1 levels and cell cycle progression was observed with 200 nM of an unrelated siRNA (si-GAPDH), or of a mismatch siRNA harbouring two mutations (Cyc-B3) complexed with MPG-8 at a 20/1 ratio, or with MPG-8 carrier alone (100 µM).Figure 2.


Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth.

Crombez L, Morris MC, Dufort S, Aldrian-Herrada G, Nguyen Q, Mc Master G, Coll JL, Heitz F, Divita G - Nucleic Acids Res. (2009)

MPG-8-mediated delivery of siRNA targeting cyclin B1 induces G2-arrest. MPG-8 (A and B) and MPG (C) dose–response of Cyclin B1 silencing at the protein and mRNA levels. Stock solutions of MPG-8/siRNA (100 nM) or MPG/siRNA (500 nM) particles were prepared at a molar ratio of 1/20, and lower concentrations (from 200 nM to 0.125 nM) were obtained by serial dilution of the stock solution in PBS. HeLa (A) and HS-68 (B and C) cells (60% confluency) were overlaid with preformed complexes for 30 min, then fresh DMEM supplemented with 10% FCS was added directly to the cells, which were then returned to the incubator for 24 h. Cyclin B1 protein levels were determined by western blotting using Cdk2 as a control for quantification (grey bars). Cyclin B1 mRNA levels were measured 12 h after transfection using Quantigen technology (white bars). Mismatched Cyc-B3 siRNA associated with MPG-8 (200 nM) and empty MPG-8 particles (20 µM) were used as a control. Dose–response of G2-arrest associated with Cyclin B1 silencing (D). HeLa (grey bars) and HS68 (white bars) cells were treated with increasing concentrations of MPG-8/siRNA-Cyc-B1 from 0.25 to 20 nM. The cell cycle status was evaluated by FACS analysis. Mismatched Cyc-B3 siRNA (100 nM) and GAPDH siRNA (100 nM) associated to MPG-8 as well as to MPG-8 carrier alone (20 µM) were used as controls. Results are the means ± of four separate experiments.
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Figure 2: MPG-8-mediated delivery of siRNA targeting cyclin B1 induces G2-arrest. MPG-8 (A and B) and MPG (C) dose–response of Cyclin B1 silencing at the protein and mRNA levels. Stock solutions of MPG-8/siRNA (100 nM) or MPG/siRNA (500 nM) particles were prepared at a molar ratio of 1/20, and lower concentrations (from 200 nM to 0.125 nM) were obtained by serial dilution of the stock solution in PBS. HeLa (A) and HS-68 (B and C) cells (60% confluency) were overlaid with preformed complexes for 30 min, then fresh DMEM supplemented with 10% FCS was added directly to the cells, which were then returned to the incubator for 24 h. Cyclin B1 protein levels were determined by western blotting using Cdk2 as a control for quantification (grey bars). Cyclin B1 mRNA levels were measured 12 h after transfection using Quantigen technology (white bars). Mismatched Cyc-B3 siRNA associated with MPG-8 (200 nM) and empty MPG-8 particles (20 µM) were used as a control. Dose–response of G2-arrest associated with Cyclin B1 silencing (D). HeLa (grey bars) and HS68 (white bars) cells were treated with increasing concentrations of MPG-8/siRNA-Cyc-B1 from 0.25 to 20 nM. The cell cycle status was evaluated by FACS analysis. Mismatched Cyc-B3 siRNA (100 nM) and GAPDH siRNA (100 nM) associated to MPG-8 as well as to MPG-8 carrier alone (20 µM) were used as controls. Results are the means ± of four separate experiments.
Mentions: Dose–response experiments performed on cultured cells revealed that MPG-8-mediated delivery of siRNA (Cyc-B1) induced a robust biological response associated with downregulation of both cyclin B1 protein and mRNA levels (Figure 2A and B). An siRNA concentration of 5 nM was sufficient to reduce cyclin B1 levels by more than 85% in HeLa cells (Figure 2A) and IC50 of 1.1 ± 0.3 nM and 0.9 ± 0.2 nM were estimated for downregulation of protein levels, and of 0.6 ± 0.1 nM and 0.4 ± 0.1 nM for mRNA levels, for non-transformed HS68 fibroblasts (Figure 2B) and HeLa cells (data not shown), respectively. In comparison, when siRNA were delivered with MPGΔNLS, IC50 values of 24 ± 5 nM and 35 ± 7 nM were obtained for downregulation of protein and mRNA levels, respectively (Figure 2C). That MPG is 30- to 60-fold less efficient than MPG-8 can be directly correlated to differences in stability, solubility and size of the siRNA/MPG complexes. Reduction of cyclin B1 protein levels was directly associated with accumulation of cells with a 4N content, consistent with downregulation of Cdk1-Cyclin B1 activity, and was optimally obtained with 2 nM siRNA and IC50 values estimated to 0.8 ± 0.2 nM and 1.2 ± 0.4 nM for HeLa and HS68 cells, respectively (Figure 2D). In contrast, no effect on cyclin B1 levels and cell cycle progression was observed with 200 nM of an unrelated siRNA (si-GAPDH), or of a mismatch siRNA harbouring two mutations (Cyc-B3) complexed with MPG-8 at a 20/1 ratio, or with MPG-8 carrier alone (100 µM).Figure 2.

Bottom Line: In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection.We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised.The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherches de Biochimie Macromoléculaire, Department of Molecular Biophysics and Therapeutic, UMR-5237 CNRS-UM2-UM1, 1919 Route de Mende, 34293 Montpellier, France.

ABSTRACT
The development of short interfering RNA (siRNA), has provided great hope for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake and bioavailability of siRNA remain a major obstacle to their clinical development and most strategies that propose to improve siRNA delivery remain limited for in vivo applications. In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection. Moreover, we show that functionalization of this carrier with cholesterol significantly improves tissue distribution and stability of siRNA in vivo, thereby enhancing the efficiency of this technology for systemic administration following intravenous injection without triggering any non-specific inflammatory response. We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised. The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.

Show MeSH
Related in: MedlinePlus