Enhancement of siRNA transfection by the optimization of fatty acid length and histidine content in the CPP
2019
Porosk, Ly | Arukuusk, Piret | Põhako, Kaisa | Kurrikoff, Kaido | Kiisholts, Kristina | Padari, Kärt | Pooga, Margus | Langel, Ülo
Extracellular synthetic nucleic acids, such as siRNAs, are unable to reach their intended targets efficiently. Therefore, delivery methods such as cell-penetrating peptides (CPP), which increase their transport, could enhance the potency of siRNA as therapeutic agents. The CPP NickFect55 (NF55) is an efficient peptide-based delivery vector, which has been previously used to deliver plasmid DNA into cells in vivo. To achieve higher intracellular delivery and bioactivity from the delivered cargo, we designed a series of histidine-containing peptides by optimizing pH-sensitivity, net charge, hydrophobicity, and charge distribution in the sequence of the CPP NF55. In the current work, we have applied a strategy where we have replaced amino acids in the C-terminus of the peptide in order to distribute hydrophobic and hydrophilic amino acids into distinct regions along the alpha-helical projection, including histidine amino acids into the sequence at the N-terminus, and optimizing the N-terminal fatty acid modification to suit the specific peptide sequence. We tested the CPPs based on the transfection efficacy, CPP/siRNA complex stability, and the properties of the CPPs, such as hemolytic activity, buffering capability and cell toxicity. As a result, we have introduced a new peptide with a completely redesigned N-terminus that displays adaptive response to its physical environment. This peptide – NickFect70 (NF70) – efficiently condenses siRNA, protects the cargo against degradation and effectively mediates target gene knockdown both in mammalian cell culture and in vivo, in a mouse model.
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