Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond

Cationic liposomes have been actively used as gene delivery vehicle because of their minimal toxicity, but their relatively low efficiency of gene delivery is the major disadvantage of these vectors. Recently, cysteine residue incorporation to HIV-1 Tat peptide increased liposomemediated transfection compared with unmodified Tat peptide. Therefore, we designed a novel modified Tat peptide having a homodimeric (Tat-CTHD, Tat-NTHD) and closed structure (cyclic Tat) simply by using the disulfide bond between cysteines to develop a more efficient and safe nonviral gene delivery system. The mixing of Tat-CTHD and Tat-NTHD with DNA before mixing with lipofectamine increased the transfection efficiency compared with unmodified Tat peptide and lipofectamine only in MCF-7 breast cancer cells and rat vascular smooth muscle cells. However, cyclic Tat did not show any improvement in the transfection efficiency. In the gel retardation assay, Tat-CTHD and Tat-NTHD showed more strong binding with DNA than unmodified Tat and cyclic Tat peptide. This enhancement was only shown when Tat-CTHD and Tat-NTHD were mixed with DNA before mixing with lipofectamine. The effects of Tat- CTHD and Tat-NTHD were also valid in the experiment using DOTAP and DMRIE instead of lipofectamine. We could not find any significant cytotoxicity in the working concentration and more usage of these peptides. In conclusion, we have designed a novel transfection-enhancing peptide by easy homodimerization of Tat peptide, and the simple mix of these novel peptides with DNA increased the gene transfer of cationic lipids more efficiently with no additional cytotoxicity.     

Proteolipidic vectors for gene transfer to the lung

In order to develop improved synthetic gene transfer vectors, we have synthesized bifunctional peptides composed of a DNA binding peptide (P2) and ligand peptides selected by the phage display technique on tracheal epithelial cells. We have evaluated the capacity of these peptides to enhance the gene transfer efficiency of the cationic lipid DOTAP to the mouse lung. To optimize the in vivo transfection efficiency, we first compared the efficiency of DOTAP to transfect the lung by either intravenous injection or aerosolization. We then tested DNA/Peptide/DOTAP complexes formed at different Peptide/DNA and DOTAP/DNA charge ratios. Under optimal conditions, precompaction of DNA by peptide P2 gave a higher expression in the mouse lung using the luciferase reporter gene than DOTAP/DNA complexes. A further increase of transfection efficiency was obtained with the bifunctional peptide P2-9. Experiments performed with the GFP reporter gene showed expression in the alveolar parenchyme.     

DNA transfer into human lung cells is improved with Tat-RGD peptide by caveoli-mediated endocytosis

Cell lines and primary cells exhibit varying degrees of resistance to DNA transfection strategies. In this study, we employed the synthetic peptide Tat-RGD (TR), composed of the HIV-1 derived translocation peptide Tat fused to the integrin binding RGD motif, as a tool for improving DNA transfer into pulmonary cells. Binding experiments between DNA and TR and cytotoxicity measurements of TR treated cells were undertaken to optimize DNA and TR concentrations for transfection. Addition of a complex of TR and DNA (TRD) to A549 cells yielded significant transgene expression. When TRD was combined with Lipofectamine (TRDL), the expression was increased by 5-fold over Lipofectamine (DL) and by approximately 30-fold over TRD-mediated transfections. Also, in primary smooth muscle cells (SMC) and fibroblasts (FB) derived from pulmonary arteries, an increase in TRDL-mediated transfection efficiency was observed by a factor of approximately 2 and approximately 3 over that of DL. Laser scanning confocal microscopy for visualizing TR-dependent DNA uptake demonstrated that the internalization of TRDL complexes is linked to caveoli in the plasma membrane. Interfering with caveoli formation by methyl-b-cyclo-dextrin drastically decreased the transfection efficiency by TR. In conclusion, the Tat-RGD peptide mediates efficient gene delivery in human pulmonary cells, in particular when combined with a standard cationic lipid based transfection reagent. The enhancement of DNA uptake by Tat-RGD is suggested to be mediated by caveoli-dependent endocytosis.     

Possible mechanism of polycation liposome (PCL)-mediated gene transfer

A novel gene transfer system utilizing polycation liposomes (PCLs), obtained by modifying liposomes with cetyl polyethylenimine (PEI), was previously developed (Gene Ther. 7 (2002) 1148). PCLs show notable transfection efficiency with low cytotoxicity. However, the mechanism of PCL-mediated gene transfer is still unclear. In this study, we examined the intracellular trafficking of PCL-DNA complexes by using HT1080 cells, fluorescent probe-labeled materials, and confocal laser scan microscopy. We found that the PCL-DNA complexes were taken up into cells by the endosomal pathway, since both cellular uptake of the complex and gene expression were blocked by wortmannin, an inhibitor of this pathway. We also observed that the plasmid DNA and cetyl PEI complex became detached from the PCL lipids and was preferentially transferred into the nucleus in the form of the complex, whereas the PCL lipids remained in the cytoplasmic area, possibly in the endosomes. In fact, nigericin, which dissipates the pH gradient across the endosomal membrane, inhibited the detachment of lipids from the PCL-DNA complex and subsequent gene expression. Taken together, our data indicate the following mechanism for gene transfer by PCLs: PCLs effectively transfer DNA to endosomes and release cetyl PEI-DNA complexes into the cytosol. Furthermore, cetyl PEI also contributes to gene entry into the nucleus.     

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 microm 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.     

Mechanism of activation of an N-ras gene in the human fibrosarcoma cell line HT1080.

A full length N-ras gene has been cloned from both the human fibrosarcoma cell line HT1080 and from normal human DNA. N-ras isolated from HT1080 will efficiently induce morphological transformation of NIH/3T3 cells in a transfection assay, whereas N-ras isolated from normal human DNA has no effect on NIH/3T3 cells. The coding regions of the normal N-ras gene have been sequenced and the predicted amino acid sequence of the N-ras product is very similar to that of the c-Ha-ras1 and c-Ki-ras2 products. By making chimeric molecules between the two cloned genes the activating alteration in the HT1080 N-ras gene has been localised to a single base change that results in an amino acid alteration at position 61 of the p21 N-ras product.     

Enhancement of gene delivery to human airway epithelial cells in vitro using a peptide from the polyoma virus protein VP1

BACKGROUND: Current liposome-based gene delivery methods for therapeutic benefit are limited by their low efficiency. One possible way to improve gene expression is to include a peptide with a nuclear localization signal (NLS) to enhance the movement of the transfection complex from the cytoplasm to the nuclei of target cells. We have tested a synthetic peptide based on the amino terminal region of the polyoma virus VP1 protein. This region has non-overlapping motifs for DNA binding and nuclear localization. METHODS: Luciferase gene transfer efficiency was evaluated using this peptide and a control peptide with a mutated NLS in subconfluent, confluent and polarized human bronchial epithelial (16HBE) cells compared to lipoplex alone. RESULTS: Gene transfer efficiency with a lipopolyplex containing the VP1 peptide enhanced gene delivery compared to lipoplex. Transfection with a lipopolyplex containing the control peptide failed to enhance gene delivery. The VP1 peptide increased the amount of plasmid associated with the nucleus while the mutant VP1 peptide did not. The order of lipopolyplex formation was important, with greatest enhancement when peptide was added to the plasmid before addition of the liposome. A bipartite peptide with the VP1 sequence and an integrin-binding motif (RGD) resulted in a reduction in gene transfer efficiency compared to lipoplex. Cell adhesion studies showed that the integrin binding associated with the RGD motif was lost when it was attached to the VP1 sequence. The combination of the two peptide sequences in cis may have compromised the function of both. CONCLUSIONS: Our results indicate that the VP1 peptide represents a strategy to enhance liposome-mediated gene delivery to airway epithelia in vitro. Comparison of transfection efficiencies between the VP1 and the mutant VP1 peptides and the direct measurement of plasmid associated with the nucleus suggests that this enhancement is caused by the NLS signal sequence in the peptide.     

Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells

We constructed multimers of the TAT-(47-57) peptide. This polycationic peptide is known to be a protein and particle transduction domain and at the same time to comprise a nuclear localization function. Here we show that oligomers of the TAT-(47-57) peptide compact plasmid DNA to nanometric particles and stabilize DNA toward nuclease degradation. At optimized vector compositions, these peptides mediated gene delivery to cells in culture 6-8-fold more efficiently than poly-L-arginine or the mutant TAT(2)-M1. When DNA was precompacted with TAT peptides and polyethyleneimine (PEI), Superfect, or LipofectAMINE was added, transfection efficiency was enhanced up to 390-fold compared with the standard vectors. As early as after 4 h of transfection, reporter gene expression mediated by TAT-containing complexes was higher than the 24-h transfection level achieved with a standard PEI transfection. When cells were cell cycle-arrested by serum starvation or aphidicolin, TAT-mediated transfection was 3-fold more efficient than a standard PEI transfection in proliferating cells. In primary nasal epithelial cells and upon intratracheal instillation in vivo, TAT-containing complexes were superior to standard PEI vectors. These data together with confocal imaging of TAT-DNA complexes in cells support the hypothesis that the TAT nuclear localization sequence function is involved in enhancing gene transfer.     

核衣壳蛋白VP39融合TAT转导肽对杆状病毒转导哺乳动物细胞的影响

为了提高昆虫杆状病毒在哺乳动物细胞中转导基因的效率,构建了重组杆状病毒AcRed-tat和AcRed。两者都能在哺乳动物细胞内表达红色荧光蛋白作为报告基因。同时,AcRed-tat带有HIV-1Tat转导肽、病毒主要衣壳蛋白基因vp39及增强型绿色荧光蛋白(egfp)三者的融合基因,并由杆状病毒多角体启动子表达,能够在昆虫细胞中表达该Tat融合蛋白,并掺入子代病毒粒子。而AcRed作为相应的对照病毒,带有多角体启动子表达vp39和egfp的融合基因。2株病毒分别转导哺乳动物细胞后,利用流式细胞仪检测报告基因的表达水平,发现在CHO和Vero细胞中AcRed-Tat介导的报告基因表达水平明显高于AcRed,而在HEK293细胞中2株病毒介导的报告基因表达水平差异不显著。结果表明Tat转导肽可以提高杆状病毒对一部分哺乳动物细胞的转导效率,为改进杆状病毒-哺乳动物细胞转导载体提供了新的思路。     

Efficient penetration of Scp01-b and its DNA transfer abilities into cells

The in vivo application potential of viral-based gene delivery approaches is hindered by a risk of insertional oncogenesis. Of the many delivery methods, cell-penetrating peptides (CPP)-based delivery has good biocompatibility and biodegradability. However, low efficiency is still the disadvantage of CPPs-based nucleic acid transfection, and delivery efficiency may vary from different CPPs. Here, we describe Scp01-b, as a new CPP, which can enter cultured cell lines and primary cultured cells examined by fluorescence microscopy and quantitative assay, the internalization process is a concentration, temperature, and incubation time-dependent manner. Scp01-b does not insert into the membrane directly and its uptake is mediated through endocytosis pathway. Moreover, Scp01-b could mediate the uptake of plasmid DNA into the Caski and HSC-T6 cells, and we noted that Scp01-b-mediated transfection efficiency was nearly the same with traditional liposome (TurboFectin)-mediated transfection. These findings suggest that Scp01-b can act as a useful tool for non-viral-based delivery in further application such as reprogramming and gene editing.     

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.     

A fish antimicrobial peptide, tilapia hepcidin TH2-3, shows potent antitumor activity against human fibrosarcoma cells

As part of a continuing search for potential anticancer drug candidates from antimicrobial peptides of marine organisms, tilapia (Oreochromis mossambicus) hepcidin TH2-3 was evaluated in several tumor cell lines. The results indicated that TH2-3, a synthetic 20-mer antimicrobial peptide, specifically inhibited human fibrosarcoma cell (HT1080 cell line) proliferation and migration. The way in which TH2-3 inhibited HT1080 cell growth was then studied. TH2-3 inhibited HT1080 cell growth in a concentration-dependent manner according to an MTT analysis, which was confirmed by a soft-agar assay and AO/EtBr staining. Scanning electron microscopy revealed that TH2-3 caused lethal membrane disruption in HT1080 cancer cells, and a wound healing assay supported that TH2-3 decreased the migration of HT1080 cells. In addition, c-Jun mRNA expression was downregulated after treatment with TH2-3 for 48–96 h compared to the untreated group. These findings suggest a mechanism of cytotoxic action of TH2-3 and indicate that TH2-3 may be a promising chemotherapeutic agent against human fibrosarcoma cells.     

Effect of transferrin as a ligand of pH-sensitive fusogenic liposome-lipoplex hybrid complexes

We previously developed potent nonviral vectors based on complexation of lipoplexes and pH-sensitive fusogenic liposomes, which achieve efficient transfection through membrane fusion with intracellular acidic compartments such as endosomes. Because transferrin receptor is known to be overexpressed in cancer cells, in this study, we investigated the effect of transferrin as a ligand for transfection of various cancer-derived cell lines mediated by the liposome-lipoplex hybrid complexes. Results showed that these hybrid complexes with transferrin exhibited higher transfection efficiency toward these cells than complexes without transferrin, but the extent of the transferrin-induced enhancement was dependent on the cell line. Conjugation of transferrin increased their transfection activity for HeLa and KB cells, although it only slightly enhanced transfection for HT1080, HepG2, and K562. Transferrin receptors in HT1080, HepG2, and K562 cells were internalized slowly, whereas those in HeLa and KB cells were internalized quickly and actively. These results indicate that transfection mediated by the ligand-attached hybrid complex does not correlate with the amount of transferrin receptor in the cell surface but correlate with the activity of internalization of transferrin receptor into the cells.     

Which mechanism for nuclear import of plasmid DNA complexed with polyethylenimine derivatives?

BACKGROUND: To investigate the nuclear import mechanism of plasmid/polyethylenimine (PEI) derivative complexes and the putative nuclear targeting of therapeutic genes by the use of oligosaccharides, we have studied the nuclear import of plasmid DNA complexed either with PEI or with lactosylated PEI (Lac-PEI) in cystic fibrosis human airway epithelial cells ( summation operatorCFTE29o- cells). METHODS AND RESULTS: Cells were synchronized by a double-thymidine block protocol and gene transfer efficiency was evaluated: Lac-PEI- and PEI-mediated gene transfer was greatly increased when cells have undergone mitosis during the course of transfection. However, both types of complexes were able to transfect some growth-arrested cells. When the nuclear import of plasmid/Lac-PEI or plasmid/unsubstituted PEI complexes was studied in digitonin-permeabilized cells, the nuclear uptake of both types of complexes did not follow the classic pathway of nuclear localization sequence (NLS)-containing proteins and lactose residues did not act as a nuclear localization signal. CONCLUSIONS: Our results show that for complexes made with PEI derivatives, the major route for plasmid DNA nuclear entry is a passive nuclear importation during mitosis when the nuclear membrane temporarily breaks down. However, albeit to a lesser extent as that observed in dividing cells, a plasmid DNA importation also occurs in nondividing cells by a yet unknown mechanism.     

Efficient in vivo gene delivery using modified Tat peptide with cationic lipids

A combination of modified HIV-1 Tat (mTat) peptide and cationic lipids, FuGENE HD (FH), dramatically enhanced transfection efficiency across a range of cell lines when compared to mTat or FH alone (Biomaterials 35:1705–1715 2014). The efficiency of this Tat peptide combination was significantly higher than many commercial non-viral vectors. In this present study, we tested the feasibility of this non-viral vector, mTat/FH, in vivo using plasmid DNA encoding a luciferase gene. The results of the in vivo studies showed that animals administered mTat/FH/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, FH/DNA, or DNA alone. Histological evaluation showed little immune response in the muscles, livers, and kidneys of mice administered with the mTat/FH. The combination of mTat with FH could significantly improve transfection efficiency, expanding the potential use of non-viral gene vectors in vivo.