The Studies On Novel Peptide-based DNA Delivery System

DNA therapy shows a promising strategy for treating various diseases but still requires efficient and safe systems that can deliver the therapeutic DNA selectively into targeted cells. Viral vectors have been used to transfer genes into cells successfully. However, these vectors have serious disadvantages, such as limited loading capacity, complexity of production, innate immunogenicity, and the risk of inflammatory responses and toxicity, that limit their clinical applications. To avoid these problems, various non-viral carriers have been developed; these carriers display low immunogenicity, relative safety, ease of production, and no cargo size limitation. Of the existing non-viral vectors, cationic lipids and cationic polymers are the most intensively studied and frequently employed. However, they have dose-dependent toxicities in in vivo applications.Cationic peptides have also been explored as gene delivery systems due to several advantages:biodegradability, biocompatibility, less toxicity, and ease of synthesis compared with polymeric carriers. Moreover, the composition of peptides is easy to control. By altering the composition of a given peptide, various functions can be achieved. However, none of these systems has any selectivity toward cells or tissues, which makes it impossible for them to deliver DNA into targeted cells or tissues in vivo and thus seriously limits their applications in gene therapy.Cell-penetrating peptides (CPPs) are one kind of short cationic peptides. In this study, we designed novel CPPs and investigated their characters. A series of heterogeneous oligoarginines consisting of natural and unnatural amino acids were synthesized. Then their uptake efficiencis, intracellular distributions and anti-enzymatic hydrolysis abilities were investigated. From the results we screened out the structure suitable for using as a DNA transport carrier. Then the target motif was added into the CPPs sequence for target DNA delivery. Firstly, the folate acid for tumor targeting was added into the chosen CPP to construct a non-viral peptide based DNA delivery system for tumor targeting DNA delivery. The results showed that although this vector could bind and compack DNA, it could not deliver DNA into the cells. This is possibly because of the steric hindrance or the low zeta potential of the peptide/DNA complex.Next, a tumor targeting sequence was added into the chosen CPP to construct a non-viral peptide-based DNA delivery system which could mediate efficient DNA expression in only selected tumor cells. The cell uptake efficiencies, intracellular distribution, uptake mechanism and cytotoxicity were analyzed. The advantages of this system include its ease of synthesis, low immunogenicity, biocompatibility, low cytotoxicity and selectivity.Finally, a brain targeting sequence was added into the chosen CPP to construct a non-viral peptide-based DNA delivery system which could mediate efficient DNA expression to mice brain after intravenous injection. The cell uptake efficiencies, intracellular distribution, uptake mechanism and cytotoxicity were analyzed. This system could mediate neuronal cell-selective DNA delivery, efficient DNA transfection to primary neurons and targeting DNA delivery into the brain. The advantages of this system include the advantages of peptide carriers mentioned above and brain targeting. The result suggest a new strategy for brain-targeting DNA delivery in vivo.