Development and Characterization of High Efficacy Cell-Penetrating Peptide via Modulation of the Histidine and Arginine Ratio for Gene Therapy.

Yu Liu,Bi-Hui Huang,Chang-Long Bi,Duo-Mei Tian,Xiao-Jun Xu,Huan-Huan Wan,Xiao-Yong Zhan,Yun-Sheng Xu,Le-Ping Yan

doi:10.3390/ma14164674

Abstract

Cell-penetrating peptides (CPPs), as non-viral gene delivery vectors, are considered with lower immunogenic response, and safer and higher gene capacity than viral systems. In our previous study, a CPP peptide called RALA (arginine rich) presented desirable transfection efficacy and owns a potential clinic use. It is believed that histidine could enhance the endosome escaping ability of CPPs, yet RALA peptide contains only one histidine in each chain. In order to develop novel superior CPPs, by using RALA as a model, we designed a series of peptides named HALA (increased histidine ratio). Both plasmid DNA (pDNA) and siRNA transfection results on three cell lines revealed that the transfection efficacy is better when histidine replacements were on the C-terminal instead of on the N-terminal, and two histidine replacements are superior to three. By investigating the mechanism of endocytosis of the pDNA nanocomplexes, we discovered that there were multiple pathways that led to the process and caveolae played the main role. During the screening, we discovered a novel peptide-HALA2 of high cellular transfection efficacy, which may act as an exciting gene delivery vector for gene therapy. Our findings also bring new insights on the development of novel robust CPPs.

Highlights

Since Friedmann and Roblin firstly proposed the concept of gene therapy in 1972, it became a promising therapeutic option with a great potential to treat many genetic and acquired diseases [1,2,3]
Several strategies have already been tested in clinical trials, and the used nucleic acids include DNA, message RNA, micro RNA, short interfering RNA, short hairpin RNA, small activating RNA and antisense oligonucleotides (ASO), and even patient-derived cellular gene therapy [4,5,6,7,8,9,10,11]
We even observed that in HALA2, HALA3 and HALA4, the N:P ratio of 1 shInotwereedstninogelyx,trwaeDeNveAn boebisnegrvveidsibthleatoinntHheAgLeAl.2M, HeAanLwAh3ilaen, dpDHNAALAn4o,ttshheoNw:Pn roantitohoef g1elshwoewreedrentaoinexedtrawDitNhiAn tbheeinwgevllissiobflethoengthele. gAesl.foMresaiRnwNhAilceo, pndDeNnAsatniootns, heoxcwenptoHn AthLeAg3el thwpareetrspeernreetseteadninsteetaddbswletaictbholinendctoehnnesdawetnieosllnastfiorofonmthfreNogm:ePlN.6A:oPsn6wfooarnrsdwiR, atNhrdeA,rtechosetnordefestnthsoeaftpitoehpnet,piedexepcsetippdrteeHssepAnrLteeAsde3nfrttohedmat N:P 4 onward (Figure 1B). This indicated that two and even three arginine replacements led to little impact on the condensational ability of the RALA peptide

Summary

Introduction

Since Friedmann and Roblin firstly proposed the concept of gene therapy in 1972, it became a promising therapeutic option with a great potential to treat many genetic and acquired diseases [1,2,3]. The primal principle of gene therapy is to introduce foreign genetic material into host cells via suitable vectors, in order to promote the expression of therapeutic proteins or to silence the relevant genes. Several strategies have already been tested in clinical trials, and the used nucleic acids include DNA, message RNA (mRNA), micro RNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), small activating RNA (saRNA) and antisense oligonucleotides (ASO), and even patient-derived cellular gene therapy [4,5,6,7,8,9,10,11]. Non-viral gene delivery systems, e.g., liposomes, cationic polymers, and cell-penetrating peptides (CPPs), are considered with properties of lower immunogenic response, safety, higher gene capacity, more stability and more flexibility of chemical design than viral systems [12]. CPPs have a variety of applications, such as acting as vectors for nucleic acid condensation, incorporation of functional motif [15,16,17], or even for anti-microbial application [18,19]

Objectives

Methods

Results

Conclusion