Development and optimization of KL4 peptide for pulmonary delivery of RNA

Abstract

Inhalation of RNA therapeutics such as siRNA and mRNA has gained considerable attention for the treatment of a wide range of respiratory diseases. The delivery of RNAs presents a major obstacle that hinders their further development. A safe and effective vector is needed to mediate robust RNA transfection in the lung. In this study, KL4 peptide and its PEGylated derivatives were investigated for pulmonary delivery of RNA. KL4 is a synthetic cationic peptide with 21 residues containing repeating KLLLL subunits. It is structurally and functionally similar to surfactant protein B (SP-B) in human pulmonary surfactant. The rationale of employing KL4 as RNA delivery vector originates from the capability of naked RNA to transfect in the lung following pulmonary delivery. This observation leads to a hypothesis that cationic endogenous components in the lung may serve as natural RNA carriers by forming complexes with the anionic RNA, thereby promoting cellular uptake. The cationic SP-B is one of the possible candidates. Instead of investigating SP-B which is structurally complex, KL4 peptide was studied as RNA vector for pulmonary delivery.

The potential of KL4 to deliver siRNA was firstly evaluated. KL4 was able to bind to siRNA and form nano-sized complexes through electrostatic interaction. KL4/siRNA complexes showed superior stability in pulmonary surfactant and they mediated effective siRNA transfection in vitro on human lung epithelial cells. There was no significant sign of inflammatory response and toxicity in vitro at the tested dose. However, the high leucine content of KL4 renders it insoluble in water and limits its application as delivery vector. To address this problem, PEGylation strategy was applied by covalently attaching monodispersed polyethylene glycol (PEG) to KL4. Three PEGs with length varied between six to 24 monomers were employed to investigate the impact of PEGylation. The increase of PEG chain length resulted in higher water solubility, improved colloidal stability and enhanced transfection efficiency in vitro. Among all the peptides, PEG12KL4 peptide, which contains 12 monomers of PEG, was identified to be optimal for siRNA delivery as it attains a balance between good solubility, efficient transfection and high safety profile.

In addition to siRNA, the potential PEG12KL4 peptide for pulmonary delivery of mRNA was further investigated. PEG12KL4 peptide can bind and form complexes with mRNA. It mediated efficient mRNA transfection both in vitro in lung epithelial cells and dendritic cells as well as in the lungs of mice following intratracheal administration. Inhalable dry powder formulations of PEG12KL4/mRNA complexes were produced using spray drying (SD) and spray freeze drying (SFD) technologies. The dry powders exhibited satisfactory stability and aerosol properties for inhalation. More importantly, the transfection efficiency of PEG12KL4/mRNA complexes was well-preserved after drying.

To conclude, KL4 and PEGylated KL4 peptides are promising candidates for delivering RNA therapeutics in the clinic. Future studies will focus on applying the delivery system to disease models to evaluate their therapeutic potential.