Gene therapy is currently a subject of great interest since it represents one of the most promising medicines for treating diseases that are not currently treatable with conventional medicines 1–3. Gene therapy relies on efficiently delivering a therapeutic nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA) to an intracellular target site for the manipulation of gene expression for therapeutic purposes. The use of replication-defective viral vectors is currently the most efficient method for delivering nucleic acids to their intracellular target sites 4,5. However, viral vectors suffer from several drawbacks including high immunogenicity, a high level of toxicity and difficulty of large-scale production. Synthetic non-viral vectors, i.e., liposomes, micelles, and dendrimers are promising alternates to viral vectors due to their simplicity, higher safety profiles and lower immunogenicity 6–9. Non-viral vectors are recently applied for treating a wide-variety of acquired and inherited diseases including cancer, cystic fibrosis, cardiovascular and neurological diseases 10–16. Despite the great progress that has been made in developing non-viral vectors in the past decade, the efficiency of such systems for use in gene therapy remains far below that of viral-based systems. Therefore, the greater challenge in non-viral gene delivery is developing systems that can overcome different extracellular and cellular barriers to efficiently and specifically deliver nucleic acids to their target sites in doses sufficient to produce a therapeutic effect 6.
Different strategies have been used for increasing the efficiency of non-viral systems including the use of novel polymers, peptides or lipids assembled in nano-delivery systems for protecting therapeutic nucleic acids and for improving their delivery to target sites inside cells 17–19. A promising strategy depends on developing systems that combine two or more functional devices that have previously proven to be efficient and that function via different mechanisms. The successful combination of such different functional devices in a single system is expected to increase the efficiency of gene delivery through synergistic effects of these devices. However, a rationalized design is required for controlling the topology of different components in a way that ensures that each one of them is functional at the right time and place.
We recently developed a pH-sensitive cationic lipid (referred to as YSK05) that is neutral at physiological pH but acquires a positive charge in acidic conditions 20. YSK05 can condense and protect pDNA or siRNA by incorporating them into stable lipid nanoparticles (NPs) owing to its positive charge when used in an acidic medium 21–25. Subsequent neutralization of the medium after the formation of the NPs produces NPs with a neutral charge, which is advantageous for systemic administration where non-specific interactions and unwanted aggregation in the circulation can be avoided. In mice, YSK05 has been shown to mediate efficient pDNA delivery, resulting in efficient gene expression in hepatocytes after in vivo administration 25. In addition, siRNA encapsulated in YSK05 NPs also resulted in efficient gene silencing in liver tissues in mice 23,24. The efficiency of YSK NPs may be related to its ability to adopt a cone shape structure in the acidic pH of endosomes, which facilitates its fusion with the endosomal membrane, thus allowing the internalized particles to escape efficiently from the endosomes 20. It, therefore, appears that YSK05 is a promising functional device for developing efficient nucleic acid-based therapeutics. YSK NPs proved to be efficient despite the expected low cellular uptake and membrane interactions of YSK NPs after the loss of their positive charge. Therefore, it would be expected that the activity of YSK-based NPs could be further improved by using other functional devices that further enhance cellular uptake and interactions with membranes in general.
On the other hand, we have previously used the octaarginine peptide (R8) for efficient gene delivery in vitro and in vivo 26–31. The positively charged R8 peptide was found to mediate efficient cellular uptake and improve the intracellular trafficking of its cargos, leading to an enhanced nuclear delivery of exogenous genes and improved gene expression 29,30. Perhaps the most important disadvantage of the R8 peptide is its permanent positive charge, which limits its activity in vivo. Our extensive research with R8 led us to conclude that this peptide can still produce a dramatic improvement in gene delivery even when its positive charge is masked and not expressed on the surface.
In this study, we hypothesized that efficient gene delivery can be achieved through the use of a combination of YSK05 and R8 in a rationally designed system. We attempted to combine YSK05 and R8 in a synergistic non-positive design based on controlling the density and topology of the peptide. The simple mixing of YSK05 and low amounts of R8 resulted in the formation of a non-positive system, albeit with low transfection activity. To improve the activities, we examined the use of a two-step coating strategy for pDNA where only the inner lipid coat is modified with a low density of R8 while the outer lipid coat is mainly composed of YSK05 for achieving efficient endosomal escape. The proposed design proved to be essential for achieving a successful synergism between YSK05 and R8. We further identified the mechanism responsible for this synergism, which indicated that the cationic R8 peptide has the ability to play multiple roles in gene delivery that extend far beyond simply the introduction of a positive charge to the system.