Peptide-based Gene Delivery Research Articles

Peptide-Based Vectors for Gene Delivery

Gene therapy is the ultimate therapeutic technology for diseases related to gene abnormality. However, the use of DNA alone has serious problems, such as poor stability and difficulty in entering target cells. The development of a safe and efficient gene delivery system is the cornerstone of gene therapy. Of particular interest, multifunctional peptides are rationally designed as non-viral vectors for efficient gene delivery. As components of gene delivery vectors, these peptides play critically important roles in skeleton construction, the implementation of targeting strategies, cell membrane penetration, endosome rupture, and nuclear transport. In recent years, the research of functional peptide-based gene delivery vectors has made important progress in improving transfection efficiency. The latest research progress and future development direction of peptide-based gene delivery vectors are reviewed in this paper.

Cell-Penetrating Peptides for Use in Development of Transgenic Plants.

Genetically modified plants and crops can contribute to remarkable increase in global food supply, with improved yield and resistance to plant diseases or insect pests. The development of biotechnology introducing exogenous nucleic acids in transgenic plants is important for plant health management. Different genetic engineering methods for DNA delivery, such as biolistic methods, Agrobacterium tumefaciens-mediated transformation, and other physicochemical methods have been developed to improve translocation across the plasma membrane and cell wall in plants. Recently, the peptide-based gene delivery system, mediated by cell-penetrating peptides (CPPs), has been regarded as a promising non-viral tool for efficient and stable gene transfection into both animal and plant cells. CPPs are short peptides with diverse sequences and functionalities, capable of agitating plasma membrane and entering cells. Here, we highlight recent research and ideas on diverse types of CPPs, which have been applied in DNA delivery in plants. Various basic, amphipathic, cyclic, and branched CPPs were designed, and modifications of functional groups were performed to enhance DNA interaction and stabilization in transgenesis. CPPs were able to carry cargoes in either a covalent or noncovalent manner and to internalize CPP/cargo complexes into cells by either direct membrane translocation or endocytosis. Importantly, subcellular targets of CPP-mediated nucleic acid delivery were reviewed. CPPs offer transfection strategies and influence transgene expression at subcellular localizations, such as in plastids, mitochondria, and the nucleus. In summary, the technology of CPP-mediated gene delivery provides a potent and useful tool to genetically modified plants and crops of the future.

Multifunctional gene delivery vectors containing different liver-targeting fragments for specifically transfecting hepatocellular carcinoma (HCC) cells.

Gene therapy is a promising strategy for HCC treatment, but it commonly faces the problem of low specificity in gene transfection. In this study, we designed and synthesized a series of peptide-based gene delivery vectors (H-01 to H-18) containing varied HCC cell-targeting fragments for specifically binding different receptors highly expressed on HCC cell membranes. The physicochemical properties of peptide vectors or peptide/DNA complexes were characterized, and the gene delivery abilities of peptide vectors were evaluated in HepG2 cell lines. The results showed that peptide vectors H-02 and H-09, which contained targeted fragments for ACE2 and c-Met receptors, respectively, could specifically transfect HCC cells in a highly -efficient manner in vitro. Furthermore, the liver-targeting function in vivo of Cy5.5 labeled H-02 (H-17) and H-09 (H-18) was investigated by fluorescence imaging.

Application of Peptides in Construction of Nonviral Vectors for Gene Delivery.

Gene therapy, which aims to cure diseases by knocking out, editing, correcting or compensating abnormal genes, provides new strategies for the treatment of tumors, genetic diseases and other diseases that are closely related to human gene abnormalities. In order to deliver genes efficiently to abnormal sites in vivo to achieve therapeutic effects, a variety of gene vectors have been designed. Among them, peptide-based vectors show superior advantages because of their ease of design, perfect biocompatibility and safety. Rationally designed peptides can carry nucleic acids into cells to perform therapeutic effects by overcoming a series of biological barriers including cellular uptake, endosomal escape, nuclear entrance and so on. Moreover, peptides can also be incorporated into other delivery systems as functional segments. In this review, we referred to the biological barriers for gene delivery in vivo and discussed several kinds of peptide-based nonviral gene vectors developed for overcoming these barriers. These vectors can deliver different types of genetic materials into targeted cells/tissues individually or in combination by having specific structure-function relationships. Based on the general review of peptide-based gene delivery systems, the current challenges and future perspectives in development of peptidic nonviral vectors for clinical applications were also put forward, with the aim of providing guidance towards the rational design and development of such systems.

Transferrin receptor targeting segment T7 containing peptide gene delivery vectors for efficient transfection of brain tumor cells

Successful gene therapy for brain tumors are often limited by two important factors, the existence of blood brain barrier (BBB) and inefficient transfection of brain tumor cells. In this study, we designed a series of peptide-based gene delivery vectors decorated with T7 segment for binding the transferrin (Tf) receptors which were highly expressed on brain tumor cells, and evaluated their ability of gene delivery. The physicochemical properties of peptide vectors or peptide/DNA complexes were studied as well. The in vitro transfection efficiency was investigated in normal and glioma cell lines. Among these complexes, PT-02/DNA complexes showed the highest transfection efficiency in glioma cells and low cytotoxicity in normal cell lines, and it could transport DNA across the BBB model in vitro. Furthermore, PT-02/DNA could deliver pIRES2-EGFP into the brain site of zebrafish in vivo. The designed peptide vectors offered a promising way for glioma gene therapy.

Dual Peptide-Based Gene Delivery System for the Efficient Transfection of Plant Callus Cells.

Owing to their diverse functions and tunable physicochemical properties, peptides are promising alternatives to the conventional gene delivery tools that are available for plant systems. However, peptide-mediated gene delivery is limited by low transfection efficiency in plants because of the insufficient cytosolic translocation of DNA cargo. Here, we report a dual peptide-based gene delivery system for the efficient transfection of plant callus cells. This system is based on the combination of an artificial peptide composed of cationic cell-penetrating and hydrophobic endosomal escape domains with a gene carrier peptide composed of amphiphilic cell-penetrating and cationic DNA-binding domains. Cellular internalization and transfection studies revealed that this dual peptide-based system enables more efficient transfection of callus cells than does a carrier peptide alone by enhancing the endocytic uptake and subsequent cytosolic translocation of a carrier peptide/DNA complex. The present strategy will expand the utility of peptide-mediated plant gene delivery for a wide range of applications and basic research.

NF-κB Inhibition Suppresses Experimental Melanoma Lung Metastasis

Background:Although novel therapeutic regimens for melanoma continue to emerge, the best current clinical response rate is still less than 60%. Moreover, antimelanoma treatments contribute to toxicities in other vital organs. In this study, we elucidate the therapeutic advantages of siRNA targeting melanoma NF-κB canonical signaling pathway with a peptide-based gene delivery nanoplex system.Methods and Results:In vitro treatment of melanoma B16-F10 cells was used to demonstrate delivery and efficacy of anti-NF-kB siRNA to cell cytoplasm with a 55 mn peptide-based gene delivery system. NF-κB (p65) knockdown was validated both at mRNA and protein levels by using RT2-PCR, western blot, and immunofluorescence cellular staining. Canonical p65 mRNA was reduced by 82% and p65 protein was reduced by 48%, which differed significantly from levels in control groups. In vivo treatment of a melanoma lung metastasis mouse model with 3-serial i.v. injections of p5RHH-p65 siRNA nanoparticles retarded growth of lung metastasis within one week by 76% (p=0.003) as compared to saline control treatments.Conclusion:Inhibition of melanoma NF-κB (p65) with systemically-delivered siRNA effectively impedes the growth and progression of experimental melanoma lung metastasis.

Nucleic acid delivery to differentiated retinal pigment epithelial cells using cell-penetrating peptide as a carrier

Nucleic acid delivery to the eye is a promising treatment strategy for many retinal disorders. In this manuscript, retinal gene delivery with non-coated and chondroitin sulphate (CS) coated amphipathic and cationic peptides was tested. The transfection and gene knockdown efficiencies were evaluated in different retinal pigment epithelial (RPE) cell models including both dividing and differentiated cells. In addition, the mobility of peptide-based gene delivery systems was examined in porcine vitreous by particle tracking analysis. The results indicate that amphipathic and cationic peptides are safe in vitro and are capable of high transgene expression and gene knockdown in dividing cells. We further demonstrate that incorporation of CS improves the efficiency of gene delivery of peptide-based systems. Most importantly, the transgene expression mediated by both non-coated and CS coated peptides was high in differentiated as well as in human primary RPE cells which are typically difficult to transfect. Coating of peptide-based gene delivery systems with CS improved diffusion in the vitreous and enhanced the stability of the polyplexes. The results indicate that a peptide-based system can be fine-tuned as a promising approach for retinal gene delivery.

Peptide-based gene delivery vectors.

Gene therapy as a strategy for disease treatment requires safe and efficient gene delivery systems that encapsulate nucleic acids and deliver them to effective sites in the cell. Due to the insecurity of viral vectors, non-viral vectors have gained great attention recently. Additional advantages of non-viral vectors include their safety, flexibility in packaging nucleic acids, and ease of production. To construct an ideal gene carrier, peptides can be incorporated into non-viral gene delivery systems as functional motifs to overcome the current barriers in gene delivery. In this review, we summarize recent developments in peptide-based gene delivery vector research.

Multifunctional peptide-lipid nanocomplexes for efficient targeted delivery of DNA and siRNA into breast cancer cells.

The identification of safe and effective delivery systems for DNA and siRNA is of great importance. Herein, we developed a well-defined, multifunctional and cell-specific lipidic peptide DEN-K(GALA)-TAT-K(STR)-CTP as a breast cancer cell targeted gene delivery vector. When combined with a lipid component (DOTAP/DOPE), the peptide/lipid hybrid system demonstrated higher gene expression or knockdown levels compared to the peptide or lipid approach alone when used to deliver pDNA or siRNA respectively, indicating synergistic enhancement of gene delivery efficiency. Importantly, this delivery strategy achieved greater knockdown of the Bcl-2 protein when compared to the leading commercial siRNA delivery system Lipofectamine® RNAiMAX, suggesting its potential utility for the targeted treatment of Bcl-2 overexpressing breast cancers.

Peptide-Based and Polypeptide-Based Gene Delivery Systems.

Peptide-based and polypeptide-based gene carriers are emerging as the most potentially useful agents in the field of gene therapy. Here we summarize the methods used for the preparation of peptides and polypeptides, address the primary types of peptide-based gene delivery systems, demonstrate their applications in gene therapy, and attempt to propose possible future directions in the development of peptide-based and polypeptide-based gene carriers for specific applications.

MPG-based nanoparticle: An efficient delivery system for enhancing the potency of DNA vaccine expressing HPV16E7

DNA vaccines against human papillomavirus (HPV) type 16 have not been successful in clinical trials, due to the lack of an appropriate delivery system. In this study, a peptide-based gene delivery system, MPG, which forms stable non-covalent nanoparticles with nucleic acids, was used for in vitro and in vivo delivery of HPV16 E7 DNA as a model antigen. The results demonstrated that at Nitrogen/Phosphate (N/P) ratio over 10:1, this peptide can effectively condense plasmid DNA into stable nanoparticles with an average size of 180–210nm and a positive surface charge. The transfection efficiency of MPG-based nanoparticles was shown to be comparable with Polyethyleneimine (PEI). The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPG-based nanoparticles as a potent delivery system in DNA vaccine formulations. Immunization with MPG/E7DNA nanoparticles at an N/P ratio of 10:1 induced a stronger Th1 cellular immune response with a predominant interferon-γ (IFN-γ) profile than those induced by E7DNA alone in a murine tumor model. These findings suggest that MPG peptide as a novel gene delivery system could have promising applications in improving HPV therapeutic vaccines.

Peptide vectors for gene delivery: from single peptides to multifunctional peptide nanocarriers.

The therapeutic use of nucleic acids relies on the availability of sophisticated delivery systems for targeted and intracellular delivery of these molecules. Such a gene delivery should possess essential characteristics to overcome several extracellular and intracellular barriers. Peptides offer an attractive platform for nonviral gene delivery, as several functional peptide classes exist capable of overcoming these barriers. However, none of these functional peptide classes contain all the essential characteristics required to overcome all of the barriers associated with successful gene delivery. Combining functional peptides into multifunctional peptide vectors will be pivotal for improving peptide-based gene delivery systems. By using combinatorial strategies and high-throughput screening, the identification of multifunctional peptide vectors will accelerate the optimization of peptide-based gene delivery systems.

Rapid and Efficient Gene Delivery into Plant Cells Using Designed Peptide Carriers

To develop a new easy and quick gene delivery system for any types of plants, we prepared ionic complexes of plasmid DNA with designed peptide carriers, each of which combined a cell-penetrating peptide (Bp100 or Tat(2)) with a polycation (nona-arginine or a copolymer of histidine and lysine). The present system via the designed peptides demonstrated rapid and efficient transient transfections into intact leaf cells of Nicotiana benthamiana and Arabidopsis thaliana without protoplast preparations. The designed peptides demonstrated significantly higher transfection efficiency in comparison to the nonfusion peptides (Bp100, Tat2, nona-arginine, and copolymer of histidine and lysine), indicating that the combination of functional peptides was a key to develop an efficient peptide-based gene delivery system. On the basis of the results, we exhibited the versatility of the designed peptide-based gene delivery system, which will explore the application of plant biotechnology.

Enhanced Nuclear Import and Transfection Efficiency of TAT Peptide-Based Gene Delivery Systems Modified by Additional Nuclear Localization Signals

Cellular uptake and nuclear localization are two major barriers in gene delivery. In order to evaluate whether additional nuclear localization signals (NLSs) can improve gene transfection efficiency, we introduced different kinds of NLSs to TAT-based gene delivery systems to form three kinds of complexes, including TAT-PV/DNA, TAT/DNA/PV, and TAT/DNA/HMGB1. The DNA binding ability of different vectors was evaluated by agarose gel electrophoresis. The in vitro transfections mediated by different complexes under different conditions were carried out. The cells treated by different complexes were observed by confocal microscopy. The MTT assay showed that all complexes did not exhibit apparent cytotoxicity in both HeLa and Cos7 cell lines even at high N/P ratios. The luciferase reporter gene expression mediated by TAT-PV/DNA complexes exhibited about 200-fold enhancement as compared with TAT/DNA complexes. Confocal study showed that, except TAT/DNA/PV, all other complexes exhibited enhanced nuclear accumulation and cellular uptake in both HeLa and Cos7 cell lines. These results indicated that the introduction of nuclear localization signals could enhance the transfection efficacy of TAT-based peptides, implying that the TAT peptide-based vectors demonstrated here have promising potential in gene delivery.

Biodistribution and blood clearance of plasmid DNA administered in arginine peptide complexes

BackgroundPeptide/DNA complexes have great potential as non-viral methods for gene delivery. Despite promising results for peptide-mediated gene delivery technology, an effective systemic peptide-based gene delivery system has not yet been developed.MethodsThis study used pCMV-Luc as a model gene to investigate the biodistribution and the in vivo efficacy of arginine peptide-mediated gene delivery by polymerase chain reaction (PCR).ResultsPlasmid DNA was detected in all organs tested 1 h after intraperitoneal administration of arginine/DNA complexes, indicating that the arginine/DNA complexes disseminated widely through the body. The plasmid was primarily detected in the spleen, kidney, and diaphragm 24 h post administration. The mRNA expression of plasmid DNA was noted in the spleen, kidney, and diaphragm for up to 2 weeks, and in the other major organs, for at least 1 week. Blood clearance studies showed that injected DNA was found in the blood as long as 6 h after injection.ConclusionsTaken together, our results demonstrated that arginine/DNA complexes are stable in blood and are effective for in vivo gene delivery. These findings suggest that intraperitoneal administration of arginine/DNA complexes is a promising tool in gene therapy.

High gene delivery in tumor by intratumoral injection of tetraarginine-PEG lipid-coated protamine/DNA

One obstacle to effective gene therapy lies in low transfection efficiency by non-viral vectors. To meet this challenge, we developed cell-penetrating peptide-based gene delivery vectors. A novel oligoarginine lipid ((Arg)n-B, n = 4, 10) conjugated to 3,5-bis(dodecyloxy)benzamide (BDB) lipid with a poly(ethylene glycol) (PEG) spacer was synthesized. Oligoarginine lipid-coated vector was prepared by the addition of (Arg)n-B to DNA/protamine complex (PD) ((Arg)n-B-PD). Transfection efficiency of (Arg)n-B-PD was compared with that of (Arg)n-B/DNA complex ((Arg)n-B/D) for in vitro and in xenograft tumor of human cervical carcinoma HeLa by intratumoral injection. Transfection efficiency in tumors and in vitro greatly depended on the charge ratios of (Arg)n-B to DNA and the length of Arg residues. In vitro, positively charged Arg10-B-PD showed the highest transfection efficiency. In contrast, in tumor transfection, negatively charged Arg4-B-PD showed the highest transfection efficiency, about 2-, 16- and 23-fold higher than PD alone, Arg10-B-PD and a commercial gene transfection reagent, respectively. This result suggests that negatively charged tetraarginine-conjugated-PEG lipid-coated PD is a promising gene delivery vector for intratumoral injection.

An insight into the gene delivery mechanism of the arginine peptide system: Role of the peptide/DNA complex size

Cationic peptides have been used successfully to transfer macromolecules into living cells. Previously, we have reported a short arginine peptide-based gene delivery system. However, the mechanisms that allow arginine peptides to promote gene delivery yet remain unknown. In the present study, we investigated the effect of the arginine peptide/DNA complex size on the transfection efficiency. After combining peptides with DNA, a 400 nm complex was observed. As the incubation time was increased, the complex grew larger, reaching 6 μm after 1 h of incubation. Transfection and cellular uptake efficiency were likewise investigated for the effects of the different sizes of complexes. Large complexes were found to be advantageous for transfection. However, better internalization efficiency was found with small complexes, indicating that the amount of peptide/DNA complexes taken up by cells is not the rate-limiting step in the final transfection efficiency. The intracellular path of the peptide/DNA complex was studied using fluorescent labeling and confocal microscopy. In the early stages of transfection, complexes were observed only on the cell surface, and these complexes migrated into cytoplasm however, after 6 h, the presence of complexes in the perinuclear region was noted. We were able to detect colocalization of green and red fluorescence in both the cytoplasm and the nucleus. These results suggest that peptide/DNA complexes reach the nucleus as associated complexes.

Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1alpha variant for local induction of angiogenesis.

Hypoxia-inducible factor (HIF) constitutes a target in therapeutic angiogenesis. HIF-1alpha functions as a sensor of hypoxia and induces expression of vascular endothelial growth factor (VEGF), which then induces angiogenesis. To explore the potential of HIF-1alpha gene therapy in stimulating wound healing, we delivered a gene encoding a stabilized form of HIF-1alpha, lacking the oxygen-sensitive degradation domain, namely HIF-1alpha deltaODD, by using a previously characterized peptide-based gene delivery vector in fibrin as a surgical matrix. The peptide vector consisted of multiple domains: (i) A cysteine-flanked lysine hexamer provided DNA interactions that were stable extracellularly but destabilized intracellularly after reduction of the formed disulfide bonds. This DNA-binding domain was fused to either (ii) a fibrin-binding peptide for entrapment within the matrix or (iii) a nuclear localization sequence for efficient nuclear targeting. The HIF-1alpha deltaODD gene was expressed and translocated to the nucleus under normoxic conditions, leading to up-regulation of vascular endothelial growth factor (VEGF)-A165 mRNA and protein levels in vitro. When the peptide-DNA nanoparticles entrapped in fibrin matrices were applied to full-thickness dermal wounds in the mouse (10 microg per wound in 30 microl of fibrin), angiogenesis was increased comparably strongly to that induced by VEGF-A165 protein (1.25 microg per wound in 30 microl of fibrin). However, the maturity of the vessels induced by HIF-1alpha deltaODD was significantly higher than that induced by VEGF-A165 protein, as shown by stabilization of the neovessels with smooth muscle. Nonviral, local administration of this potent angiogenesis-inducing gene by using this peptide vector represents a powerful approach in tissue engineering and therapeutic angiogenesis.

198. Design and Testing of Intrinsic Proteasome Inhibitors To Increase Gene Transfer Efficiency

The proteasome is a multisubunit complex that is responsible for the degradation of many cytosolic proteins. Unlike many other proteolytic enzymes, the proteasome has multiple peptidase activities that can be classified into three main groups: cleavage after hydrophobic side chains (chymotrypsin-like), cleavage after basic residues (trypsin-like), and cleavage after acidic residues (peptidylglutamyl peptide hydrolysis or PGPH). Previous studies have shown that the proteasome is a key route of metabolism for peptide-based non-viral gene delivery systems 1. The gene transfer efficiency of peptide-DNA condensates can be enhanced by the addition of proteasome inhibitors, including MG115, MG132, or epoxomicin.