The therapeutic use of nucleic acids is based on the availability of sophisticated delivery systems for the targeted and intracellular delivery of these molecules. Such gene delivery should possess essential characteristics to overcome various extracellular and intracellular barriers. Peptides offer an attractive platform for the delivery of non-viral genes, as there are several classes of functional peptides capable of overcoming these barriers. However, none of these functional peptide classes contains all the essential characteristics necessary to overcome all the barriers associated with successful gene delivery. Combining functional peptides into multifunctional peptide vectors will be critical to improving peptide-based gene delivery systems. Through the use of combinatorial strategies and high-throughput screening, the identification of multifunctional peptide vectors will accelerate the optimization of peptide-based gene delivery systems.
The gene delivery system must be able to (A) tightly condense the nucleic acid charge (NA charge) into nano-sized particles and protect it against degradation; binding (B) specifically or (C) nonspecifically to the intended target cells; (D) facilitate direct cell entry or (E) internalize and induce escape of endosomolytic vesicles; and in case of nuclear supply (F), bring the nucleic acid charge to the nucleus.
Figure 2. The proposed random integrator approach that selects optimal combinations of functional peptides for efficient gene transfer by combinatorial protein engineering.
Functional domains (DCP, CPP, NLS peptides, and TP) can be randomly combined at the genetic level to generate a combinatorial gene library that encodes multimodular peptide vectors. The gene library can then be inserted into an expression vector and expressed in a suitable host organism. After subsequent expression and high-throughput purification, multimodular peptide vectors can be directly screened and the most efficient candidates selected.
CPP: cell penetrating peptide; DCP: DNA condensation peptide; NLS: nuclear localization signal; TP: target peptide.