The histidine-rich peptide LAH4-L1 strongly promotes PAMAM-mediated transfection at low nitrogen to phosphorus ratios in the presence of serum

Burkhard Bechinger,Nan Liu,Regine Süss

doi:10.1038/s41598-017-10049-y

Abstract

Non-viral vectors are widely used and investigated for the delivery of genetic material into cells. However, gene delivery barriers like lysosomal degradation, serum inhibition and transient gene expression so far still limit their clinical applications. Aiming to overcome these limitations, a pH-sensitive hybrid gene vector (PSL complex) was designed by self-assembly of poly(amidoamine) (PAMAM) dendrimers, the histidine-rich peptide LAH4-L1 and the sleeping beauty transposon system (SB transposon system, a plasmid system capable of efficient and precise genomic insertion). Transfection studies revealed that PSL complexes achieved excellent efficiency in all investigated cell lines (higher than 90% in HeLa cells and over 30% in MDCK cells, a difficult-to-transfect cell line). Additionally, the PSL complexes showed high serum tolerance and exhibited outstanding transfection efficiency even in medium containing 50% serum (higher than 90% in HeLa cells). Moreover, a high level of long-term gene expression (over 30% in HeLa cells) was observed. Furthermore, PSL complexes not only resulted in high endocytosis, but also showed enhanced ability of endosomal escape compared to PAMAM/DNA complexes. These results demonstrate that simple association of PAMAM dendrimers, LAH4-L1 peptides and the SB transposon system by self-assembly is a general and promising strategy for efficient and safe gene delivery.

Highlights

Gene therapy is a promising therapeutic strategy for diseases caused by genetic defects and, a variety of gene delivery systems have been developed[1, 2]
The LAH4-L1 peptide is positively charged at physiologic pH due to the hydrophilic surface in aqueous solution, which is a favor for binding and condensation of nucleic acids, whereas its hydrophobic surface is suitable for efficient membrane interactions[12, 13]
Addition of PAMAM reversed surface charge from negative to positive, suggesting that PAMAM could bind to the surface of SB transposon system (SBTS)/LAH4-L1 complexes

Summary

Introduction

Gene therapy is a promising therapeutic strategy for diseases caused by genetic defects and, a variety of gene delivery systems have been developed[1, 2]. PAMAM dendrimer possess a unique surface of primary amino groups which associate plasmid DNA or siRNA7 via electrostatic interaction, resulting in the formation of nanoscaled complexes, which facilitate cellular uptake and aid in the endosomal escape by the “proton sponge effect”[8]. Similar to cationic PAMAM dendrimers, the LAH4-L1 peptide spontaneously forms complexes with DNA or siRNA but uses a different strategy for endosomal escape. It promotes endosomal escape by “endosomal destabilization effect” owing to its ability to destabilize the endosomal membrane when the four histidine residues are positively charged at low pH14, 15. The SBTS requires a delivery system to effectively enter a cell[18, 19]

Methods

Results

Discussion

Conclusion