Development of A2G80 peptide-gene complex for targeted ...

Abstract

Therapeutic strategies based on antisense oligonucleotides and therapeutic genes are being extensively investigated for the treatment of hereditary muscle diseases and hold great promise. However, the cellular uptake of these polyanions to the muscle cells is inefficient. Therefore, it is necessary to develop more effective methods of gene delivery into the muscle tissue. The A2G80 peptide (VQLRNGFPYFSY) from the laminin α2 chain has high affinity for α-dystroglycan (α-DG) which is expressed on the membrane of muscle cells. In this study, we designed a peptide-modified A2G80 with oligoarginine and oligohistidine (A2G80-R9-H8), and prepared peptide/plasmid DNA (pDNA) complex, to develop an efficient gene delivery system for the muscle tissue. The peptide/pDNA complex showed α-DG-dependent cellular uptake of the A2G80 sequence and significantly improved gene transfection efficiency mediated by the oligohistidine sequence in C2C12 myoblast cells. Further, the peptide/pDNA complex promoted efficient and sustained gene expression in the Duchenne muscular dystrophy mouse models. The A2G80-R9-H8 peptide has the potential for use as a specific carrier for targeting muscle in gene therapy in muscular dystrophy.

Introduction

Gene therapy has made huge progress in the past few decades and various gene therapy products are being widely used in the treatment of inherited and acquired diseases. Muscular dystrophy is a hereditary disease characterized by weakness and atrophy of muscles due to degeneration of the muscle fibers. Duchenne muscular dystrophy (DMD) is one of the most severe forms of muscular dystrophy and has a high incidence rate with an estimated incidence rate is 1 in 3600 male infants [1]. DMD patients have mutations in the gene encoding the dystrophin protein which contributes to the mechanical strength of muscle tissue [2,3], leading to the loss of muscle function. It is therefore important to restore dystrophin expression in the skeletal, cardiac, and respiratory muscles to restore the muscle functionality. Recently, antisense-oligonucleotide-induced exon skipping therapy such as phosphorodiamidate morpholino oligonucleotide (PMO), has been extensively researched for application in DMD [4], and in 2016, Eteprilsen, an antisense oligonucleotide, was the first drug approved for the treatment of DMD [5]. However, the high renal clearance rate and low accumulation ratio of the antisense oligonucleotides in the muscle tissue result in low overall availability in the tissue, and there is scope for improving the efficiency of their delivery [6]. Further, safer and more effective strategies based on non-viral vectors to improve the transfection efficiency of these oligonucleotides into muscle cells are needed, because viral vectors carry the inherent risk of toxicity and immunogenicity [7,8]. Therefore, there is an urgent need to develop novel gene delivery systems (GDSs) for high efficacy gene transfection and tissue selectivity.

Multiple transfection approaches have been developed for gene delivery, including cationic lipids and cationic polymers. Cationic peptides form a complex with the negatively charged gene to improve its stability against nucleases, and deliver the gene by promoting cellular uptake [9]. However, polycation/gene complexes lack tissue selectivity and tend to be cytotoxic due to the highly charged particle surface [10]. In addition, for efficient gene transfection, it is important to improve the escape of the nanoparticles from endosomes following uptake into the cell. To promote endosomal escape of nanoparticles, many studies have focused on designing pH-responsive structures which enable the nanoparticles to escape from the endosomes through proton sponge effect [11]. In recent years, gene transfection methods using peptide-carriers have also been widely studied. Compared to cationic lipids or polymers, peptides are more biocompatible and easier to design with multiple functionalities including specific receptor targeting, improved cell permeability, and intracellular distribution. The cell-penetrating peptide (CPP), oligoarginine, has been reported to improve membrane permeability [12]. Oligoarginine has been also used to form a complex with polyanionic genes through electrostatic interactions [13]. To improve intracellular distribution, oligohistidine have been studied as a part of the sequence of a multifunctional peptide carrier to promote endosomal escape based on the proton sponge effect in cells [14,15]. However, there are limited reports on a peptide that is selective for muscle tissues.

Laminin, a major component of the basement membrane, interacts with α-dystroglycan (α-DG) and plays a critical biological role in the muscle. Previously, we screened α-DG-binding peptides using several synthetic laminin peptides and identified A2G80 (VQLRNGFPYFSY) in the laminin α2 chain G domain, which is known to be an α-DG-binding domain and the absence of the binding induces muscle diseases such as muscular dystrophy [16,17].

In this study, we focused on the A2G80 peptide and designed a muscle tissue specific peptide carrier using oligoarginine and oligohistidine to achieve formation of a complex with the plasmid DNA (pDNA). The peptide/pDNA complexes were evaluated for their cellular uptake and endosomal escape in vitro. We also examined their in vivo gene transfection efficiency and gene expression pattern using an in vivo imaging system.

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Materials

pcDNA3-Luc plasmid, derived from pGL3-basic (Promega, Madison, WI), was used as a pDNA encoding the firefly luciferase gene under the control of a cytomegalovirus (CMV) enhancer-promoter. pEGFP-N3 is an expression vector encoding the enhanced-green fluorescent protein derived from Aequorea Victoria under the control of a CMV enhancer-promoter (Clontech Laboratories Inc., Mountain View, CA). GelRed was purchased from Biotium (Fremont, CA). YOYO-1 Iodide was purchased from Thermo Fisher

Design of peptide-carriers using a laminin peptide and cationic oligomers

Two peptide-carriers were designed for muscle tissue-specific gene delivery. Although A2G80 has a high affinity for α-DG, it contains a cationic amino acid rendering it difficult to form a complex with DNA. Oligoarginine was developed as a CPP [12] that enabled the formation of a peptide/gene complex through electrostatic interactions. We combined A2G80 with oligoarginine (R9; as previously reported [12,13]) and prepared A2G80-R9 (Table 1). Additionally, to improve the intracellular

Conclusion

In conclusion, A2G80-R9 has dystroglycan-targeting ability and increases the delivery of the pDNA into muscle cells through the endosomal pathway. Furthermore, addition of oligohistidine to A2G80-R9 improved the intracellular distribution of the pDNA and enhanced the gene transfection efficiency. Efficient gene transfection activity of A2G80-R9-H8 was also demonstrated through in vivo experiments. The A2G80-R9-H8 peptide may be a useful gene delivery tool for muscle targeting. Our results

Declaration of Competing Interest

The authors declare no other competing interests.

Acknowledgements

This research was supported by the MEXT-supported Program for the Strategic Research Foundation at Private Universities. We thank Drs. Motoi Kanagawa, Tatsushi Toda, and Kazuhiro Kobayashi for their kind gift of pDNA encoding recombinant α-DG.

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