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Published on: April 16, 2019
Fusogenic peptides enhance endosomal escape improving siRNA-induced silencing of oncogenes
Sabrina Oliveira1, Inge van Rooy, Onno Kranenburg
1Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80.082, 3508 TB Utrecht, The Netherlands. S.Oliveira@pharm.uu.nl
Abstract:
Small interfering RNA (siRNA) molecules are the functional mediators of a post-transcriptional gene silencing process known as RNA interference (RNAi). The silencing of genes involved in diseases, using siRNA, is considered a very promising therapeutic strategy. However, as for all the nucleic acid based therapeutics, these negatively charged and hydrophilic molecules do not readily cross biological membranes. The use of cationic carriers generally results in positively charged complexes which are taken up by cells through endocytosis. Still, for gene silencing, these complexes need to escape through the endosomal membrane, thereby reaching the cytosol where all the RNAi machinery is present. One of the strategies developed to facilitate endosomal escape mimics the fusion of viral envelopes with host cell endosomal membranes, which occurs during viral infections. Several synthetic fusogenic peptides have been synthesized based on the fusion domain of the influenza virus. In this study we evaluated the effects of the influenza-derived fusogenic peptide diINF-7 on gene silencing efficiency of siRNA targeting the epidermal growth factor receptor (EGFR) and the K-ras oncogenes. For both targets, strong enhancement of gene silencing activity was noted after addition of diINF-7 fusogenic peptide, identifying endosomal escape as a limiting factor for siRNA silencing efficiency.
Insights
Small interfering RNA (siRNA) effectively silences genes but struggles to enter cells. Adding a fusogenic peptide, diINF-7, significantly enhances siRNA gene silencing by aiding endosomal escape.
Area of Science:
- Molecular Biology
- Biochemistry
- Drug Delivery
Background:
- Small interfering RNA (siRNA) mediates RNA interference (RNAi) for gene silencing, a promising therapeutic strategy.
- siRNA's therapeutic potential is limited by its inability to cross biological membranes due to its negative charge.
- Cellular uptake of siRNA via cationic carriers leads to endocytosis, requiring endosomal escape for cytosolic access and RNAi machinery engagement.
Purpose of the Study:
- To evaluate the efficacy of the influenza-derived fusogenic peptide diINF-7 in enhancing siRNA gene silencing.
- To investigate the role of endosomal escape as a limiting factor in siRNA-mediated gene silencing.
- To assess diINF-7's impact on silencing genes targeted by siRNA, specifically EGFR and K-ras.
Main Methods:
- Utilized synthetic fusogenic peptide diINF-7, based on the influenza virus fusion domain.
- Tested diINF-7 in combination with siRNA targeting the epidermal growth factor receptor (EGFR) and K-ras oncogenes.
- Quantified gene silencing efficiency in the presence and absence of the fusogenic peptide.
Main Results:
- Addition of diINF-7 significantly enhanced gene silencing activity for both EGFR and K-ras targets.
- Observed a strong improvement in siRNA efficacy when combined with the diINF-7 fusogenic peptide.
- Results indicate that endosomal escape is a critical bottleneck for effective siRNA gene silencing.
Conclusions:
- The influenza-derived fusogenic peptide diINF-7 effectively enhances siRNA gene silencing.
- Facilitating endosomal escape is crucial for improving the efficiency of nucleic acid-based therapeutics like siRNA.
- This strategy holds potential for advancing RNAi-based therapies by overcoming cellular delivery barriers.
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