Enhancement of phage-mediated gene transfer by nuclear localization signal

The cell membrane and the nuclear membrane are two major barriers hindering the free movement of various macromolecules through animal cells. Nevertheless, some proteins can actively bypass these barriers by dint of intrinsic peptidic signals, so incorporation of these signals might improve the efficacy of artificial gene delivery vehicles. We examined the role of the nuclear localization signal (NLS) in gene transfer, using recombinant lambda phage as a model of the polymer/DNA complexes. We prepared a lambda phage displaying a 32-mer NLS of SV40 T antigen on its surface (NLS phage), and found that this NLS phage, delivered into the cytoplasm by appropriate devices, has higher affinity for the nucleus and induces the expression of encapsulated marker genes more efficiently than does the wild-type phage. This suggests that the 32-mer NLS peptide will become a practical tool for artificial gene delivery vehicles with enhanced nuclear targeting activity.     

In vivo gene delivery and expression by bacteriophage lambda vectors

AIMS: Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo. METHODS AND RESULTS: Mice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer. CONCLUSIONS: Unmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up -- at least in part -- by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.     

A novel peptide, THALWHT, for the targeting of human airway epithelia

Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer as the major problem confronting cystic fibrosis gene therapy. To elucidate novel ligands targeting abundant, apically located receptors on airway epithelial cells, a phage display library was screened for peptides binding with high affinity to such cells. This screening yielded a selectively enriched amino acid sequence, Thr-His-Ala-Leu-Trp-His-Thr (THALWHT). Subsequent binding studies confirmed that THALWHT-displaying phages bound much stronger than phages displaying control peptides to human airway epithelial cells. In contrast, no significant binding differences were observed on a variety of non-airway-derived human cell lines suggesting selective binding of the THALWHT motif to airway epithelia. Confocal microscopy of such cells after exposure to labelled synthetic THALWHT peptide indicated that its binding is followed by specific internalisation via endocytosis. A synthetic peptide comprising a cyclic CTHALWHTC domain and a DNA binding moiety enabled efficient targeted gene delivery into human airway epithelial cells. Competition assays with free THALWHT peptide confirmed the specificity of gene delivery. Thus, the THALWHT motif may prove a useful targeting moiety for both non-viral and viral gene therapy vectors.     

Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage.

We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.     

Recombinant λ-phage nanobioparticles for tumor therapy in mice models

Lambda phages have considerable potential as gene delivery vehicles due to their genetic tractability, low cost, safety and physical characteristics in comparison to other nanocarriers and gene porters. Little is known concerning lambda phage-mediated gene transfer and expression in mammalian hosts. We therefore performed experiments to evaluate lambda-ZAP bacteriophage-mediated gene transfer and expression in vitro. For this purpose, we constructed recombinant λ-phage nanobioparticles containing a mammalian expression cassette encoding enhanced green fluorescent protein (EGFP) and E7 gene of human papillomavirus type 16 (λ-HPV-16 E7) using Lambda ZAP- CMV XR vector. Four cell lines (COS-7, CHO, TC-1 and HEK-239) were transduced with the nanobioparticles. We also characterized the therapeutic anti-tumor effects of the recombinant λ-HPV-16 E7 phage in C57BL/6 tumor mice model as a cancer vaccine. Obtained results showed that delivery and expression of these genes in fibroblastic cells (COS-7 and CHO) are more efficient than epithelial cells (TC-1 and HEK-239) using these nanobioparticles. Despite the same phage M.O.I entry, the internalizing titers of COS-7 and CHO cells were more than TC-1 and HEK-293 cells, respectively. Mice vaccinated with λ-HPV-16 E7 are able to generate potent therapeutic antitumor effects against challenge with E7- expressing tumor cell line, TC-1 compared to group treated with the wild phage. The results demonstrated that the recombinant λ-phages, due to their capabilities in transducing mammalian cells, can also be considered in design and construction of novel and safe phage-based nanomedicines.     

Evidence for a plasma membrane-mediated permeability barrier to Tat basic domain in well-differentiated epithelial cells: lack of correlation with heparan sulfate

Membrane permeation peptides, such as Tat basic domain, have emerged as useful membrane transduction agents with potential utility in therapeutic delivery and diagnostic imaging. While generally thought to universally permeate all cells by a nonselective process, the mechanism of membrane transduction remains poorly characterized. To examine vectorial transport properties of Tat basic domain in well-differentiated epithelial cells possessing tight junctions, L and D stereoisomers of Tat(48-57) peptide conjugates labeled with (99m)Tc were quantitatively analyzed in confluent monolayers of MDCK renal epithelial and CaCo-2 colonic carcinoma cells grown in transwell configurations. In both cell lines, vectorial transepithelial apparent permeability coefficients (P(app)) for L- and D-[(99m)Tc]Tat-peptides ranged from 30 to 70 nm/s, comparable to values for the macromolecular impermeant marker inulin in both apical-to-basolateral and basolateral-to-apical directions, but 100-fold less than the P(app) values for propranolol, a highly permeable control compound. Upon direct instillation of [(99m)Tc]Tat-peptide into the urinary bladder of living rats in vivo, no transepithelial permeation into other tissues was identified. Furthermore, MDCK and CaCo-2 cells showed a complete lack of intracellular accumulation of fluorescein conjugated Tat-peptide. However, translocation into cells was induced by treatment with plasma membrane permeabilizing agents such as digitonin and acetone/methanol, while cholesterol depletion with beta-methyl-cyclodextrin and metabolic inhibition with CCCP or 4 degrees C showed no effect. By contrast, in Hela and KB 3-1 cells, epithelial lines that do not form tight junctions in monolayer culture, baseline cytoplasmic and nucleolar accumulation was readily observed. Because all four cell lines expressed heparan sulfate proteoglycans, putative receptors for Tat basic peptides, we found no correlation between heparan sulfate and the permeation barrier observed in MDCK and CaCo-2 cells. The unanticipated presence of a permeation barrier to Tat-peptides in well-differentiated epithelial cells suggests the existence of cell-specific mechanisms for mediated translocation of these permeation peptides.     

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.     

Tat碱性结构域-Bcl-Xs融合蛋白与细胞短暂共培养可诱导细胞程序性死亡

艾滋病毒的 Tat蛋白碱性结构域的 1 3个氨基酸的多肽能自主穿越细胞膜并将与之融合表达的其他蛋白带入细胞 .为探讨融合表达的蛋白是否可保持原有的主要生物学功能 ,将 Bcl- Xs蛋白与 Tat碱性结构域在大肠杆菌中融合表达 ,重组蛋白 Tat- Bcl- Xs与 CHO、CNE、Hela和 772 1细胞共温育一段时间后 ,细胞均可见明显生长抑制 ,同时细胞变圆、变小、细胞核收缩 ,并有染色体凝聚、边缘化 ,或细胞核分裂为小核的现象 ,同时出现梯状 DNA,为较典型的细胞程序性死亡 ( PCD)表现 .同时发现放线菌素 D、雷公藤内脂醇与 Tat- Bcl- Xs在诱导细胞 PCD时存在交互作用 .提示Tat- Bcl- Xs蛋白可自主进入细胞内 ,并保持了 Bcl- Xs蛋白的 PCD诱导作用 ,初步证实了利用 Tat碱性结构域进行药物设计的可行性 ,同时也提示 Tat- Bcl- Xs蛋白可能可作为多种药物联合治疗中的一员而在抗肿瘤治疗中发挥作用     

Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells.

Although phage display is a powerful way of selecting ligands against purified target proteins, it is less effective for selecting functional ligands for complex targets like living cells. Accordingly, phage display has had limited utility in the development of targeting agents for gene therapy vectors. By adapting a filamentous bacteriophage for gene delivery to mammalian cells, however, we show here that it is possible to screen phage libraries for functional ligands capable of delivering DNA to cells. For example, when targeted with epidermal growth factor (EGF), M13 bacteriophage were capable of delivering a green fluorescent protein (GFP) gene to EGF receptor bearing cells in a ligand-, time-, and phage concentration-dependent manner. The EGF-targeted phage transduced COS-1 cells in a highly specific manner as demonstrated by competition with excess free EGF or alternatively with anti-EGF receptor antibodies. We further demonstrate that EGF-phage can be selected, by their ability to transduce EGF receptor bearing cells from libraries of peptide display phage. When phage were incubated with COS-1 cells, EGF ligand-encoding sequences were recovered by PCR from FACsorted, GFP-positive cells and the EGF-displaying phage were enriched 1 million-fold by four rounds of selection. These data suggest the feasibility of applying molecular evolution to phage gene delivery to select novel cell-specific DNA-targeting ligands. The same approach could be used to select genetically altered phage that are specifically designed and evolved as gene therapy vectors.     

Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.     

Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression

A recombinant, multifunctional protein has been designed for optimized, cell-targeted DNA delivery and gene expression in mammalian cells. This hybrid construct comprises a viral peptide ligand for integrin alpha(V)beta(3) binding, a DNA-condensing poly-L-lysine domain, and a complete, functional beta-galactosidase protein that serves simultaneously as purification tag and DNA-shielding agent. This recombinant protein is stable; it has been produced successfully in Escherichia coli and can be purified in a single step by affinity chromatography. At optimal molar ratios, mixtures of this vector and a luciferase-reporter plasmid form stable complexes that transfect cultured cells. After exposure to these cell-targeted complexes, steady levels of gene expression are observed for more than 3 days after transfection, representing between 20 and 40% of those achieved with untargeted, lipid-based DNA-condensing agents. The principle to include viral motifs for cell infection in single polypeptide recombinant proteins represents a promising approach towards the design of non-viral modular DNA transfer vectors that conserve the cell-target- ing specificity of native viruses and that do not need further processing after bioproduction in a recombinant host.     

A tractable method for simultaneous modifications to the head and tail of bacteriophage lambda and its application to enhancing phage-mediated gene delivery

There is considerable interest in the use of bacteriophage vectors for mammalian cell gene transfer applications, due to their stability, excellent safety profile and inexpensive mass production. However, to date, phage vectors have been plagued by mediocre performance as gene transfer agents. This may reflect the complexity of the viral infection process in mammalian cells and the need to refine each step of this process in order to arrive at an optimal, phage-based gene transfer system. Therefore, a flexible system was designed that alowed for the introduction of multiple modifications on the surface of bacteriophage lambda. Using this novel method, multiple peptides were displayed simultaneously from both the phage head and tail. Surface head display of an ubiquitinylation motif greatly increased the efficiency of phage-mediated gene transfer in a murine macrophage cell line. Gene transfer was further increased when this peptide was displayed in combination with a tail-displayed CD40-binding motif. Overall, this work provides a novel system that can be used to rationally improve bacteriophage gene transfer vectors and shows it may be possible to enhance the efficiency of phage-mediated gene transfer by targeting and optimizing multiple steps within the viral infection pathway.     

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。     

Chain length of cationic alpha-helical peptide sufficient for gene delivery into cells.

To define the minimal peptide length needed for gene delivery into mammalian cells, we synthesized several peptides with shortened chain lengths from the amino-termini of the original amphiphilic peptides (4(6), Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH( 2,) and Hel 11-7, KLLK-LLLK-LWKK-LLKL-LK), which have been known to have gene transfer abilities into cells. Each synthetic peptide was studied for its ability to bind and aggregate with plasmid DNA and the structural change of the peptide caused by binding with the DNA to establish a relative in vitro gene transfection efficiency in COS-7 cells. As a result, the deletion of eight amino acid residues of 4(6) had little influence on their ability, whereas that of 12 amino acid residues remarkably reduced the abilities to make aggregates and transfer the DNA into the cell. In the case of the Hel 11-7 series peptides, deletion of amino acid residues caused a considerable reduction in abilities to bind and form aggregates with DNA and to transfer the DNA into cell in due order. In summary, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA and transfer the DNA into the cells in the deletion series of 4(6) and Hel 11-7, respectively. Furthermore, it was indicated that reduction of membrane perturbation activity of the peptide-DNA complex due to deletion of the peptide chain length caused suppression of the transfection efficiency even if the complex was incorporated into the cells. Transfer of the complex to cytosol mediated by membrane perturbation activity of the peptide is an important step for efficient protein expression from its cDNA. The results of this study will make it easy to design and synthesize a functional gene carrier molecule such as a carbohydrate-modified peptide used in targeted gene delivery.     

HIV-1 transactivator protein Tat induces proliferation and TGF beta expression in human articular chondrocytes

The human immunodeficiency virus-1 (HIV-1) protein Tat binds to cell surface antigens and can regulate cellular responses. Tat has similar immunosuppressive effects as transforming growth factor-beta (TGF beta) and both inhibit lymphocyte proliferation. TGF beta is expressed by primary human articular chondrocytes and is their most potent growth factor. The present study analyzed the interactions of TGF beta and HIV Tat in the regulation of human articular chondrocytes. Synthetic or recombinant full-length Tat (1-86) induced chondrocyte proliferation and this was of similar magnitude as the response to TGF beta. Tat peptides that did not contain the RGD motif had similar chondrocyte stimulatory activity as full-length Tat. Among a series of Tat peptides, peptide 38-62 which contains the basic domain was the only one active, suggesting that this region is responsible for the effects on chondrocyte proliferation. Full-length Tat and peptide 38-62 synergized with TGF beta and induced proliferative responses that were greater than those obtained with any combination of the known chondrocyte growth factors. Further characterization of the interactions between Tat and TGF beta showed that Tat increased synthesis and TGF beta activity and TGF beta 1 mRNA levels. The stimulatory effects of Tat and peptide 38-62 on chondrocyte proliferation were reduced by neutralizing antibodies to TGF beta and by TGF beta antisense oligonucleotides. These results identify a virally encoded protein and a synthetic peptide derived from it as novel and potent chondrocyte growth stimuli which act at least in part through the induction of TGF beta.     

Membrane fusion activity of purified SipB, a Salmonella surface protein essential for mammalian cell invasion

An early event in Salmonella infection is the invasion of non-phagocytic intestinal epithelial cells. The pathogen is taken up by macropinocytosis, induced by contact-dependent delivery of bacterial proteins that subvert signalling pathways and promote cytoskeletal rearrangement. SipB, a Salmonella protein required for delivery and invasion, was shown to localize to the cell surface of bacteria invading mammalian target cells and to fractionate with outer membrane proteins. To investigate the properties of SipB, we purified the native full-length protein following expression in recombinant Escherichia coli. Purified SipB assembled into hexamers via an N-terminal protease-resistant domain predicted to form a trimeric coiled coil, reminiscent of viral envelope proteins that direct homotypic membrane fusion. The SipB protein integrated into both mammalian cell membranes and phospholipid vesicles without disturbing bilayer integrity, and it induced liposomal fusion that was optimal at neutral pH and influenced by membrane lipid composition. SipB directed heterotypic fusion, allowing delivery of contents from E. coli-derived liposomes into the cytosol of living mammalian cells.     

Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans

Tat, the transactivator protein of human immunodeficiency virus-1, has the unusual capacity of being internalized by cells when present in the extracellular milieu. This property can be exploited for the cellular delivery of heterologous proteins fused to Tat both in cell culture and in living animals. Here we provide genetic and biochemical evidence that cell membrane heparan sulfate (HS) proteoglycans act as receptors for extracellular Tat uptake. Cells genetically defective in the biosynthesis of fully sulfated HS are selectively impaired in the internalization of recombinant Tat fused to the green fluorescent protein, as evaluated by both flow cytometry and functional assays. In wild type cells, Tat uptake is competitively inhibited by soluble heparin and by treatment with glycosaminoglycan lyases specifically degrading HS chains. Cell surface HS proteoglycans also mediate physiological internalization of Tat green fluorescent protein released from neighboring producing cells. In contrast to extracellular Tat uptake, both wild type cells and cells genetically impaired in proteoglycan synthesis are equally proficient in the extracellular release of Tat, thus indicating that proteoglycans are not required for this process. The ubiquitous distribution of HS proteoglycans is consistent with the efficient intracellular delivery of heterologous proteins fused with Tat to different mammalian cell types.     

A Novel Peptide Enhances Therapeutic Efficacy of Liposomal Anti-Cancer Drugs in Mice Models of Human Lung Cancer

Lung cancer is the leading cause of cancer-related mortality worldwide. The lack of tumor specificity remains a major drawback for effective chemotherapies and results in dose-limiting toxicities. However, a ligand-mediated drug delivery system should be able to render chemotherapy more specific to tumor cells and less toxic to normal tissues. In this study, we isolated a novel peptide ligand from a phage-displayed peptide library that bound to non-small cell lung cancer (NSCLC) cell lines. The targeting phage bound to several NSCLC cell lines but not to normal cells. Both the targeting phage and the synthetic peptide recognized the surgical specimens of NSCLC with a positive rate of 75% (27 of 36 specimens). In severe combined immunodeficiency (SCID) mice bearing NSCLC xenografts, the targeting phage specifically bound to tumor masses. The tumor homing ability of the targeting phage was inhibited by the cognate synthetic peptide, but not by a control or a WTY-mutated peptide. When the targeting peptide was coupled to liposomes carrying doxorubicin or vinorelbine, the therapeutic index of the chemotherapeutic agents and the survival rates of mice with human lung cancer xenografts markedly increased. Furthermore, the targeting liposomes increased drug accumulation in tumor tissues by 5.7-fold compared with free drugs and enhanced cancer cell apoptosis resulting from a higher concentration of bioavailable doxorubicin. The current study suggests that this tumor-specific peptide may be used to create chemotherapies specifically targeting tumor cells in the treatment of NSCLC and to design targeted gene transfer vectors or it may be used one in the diagnosis of this malignancy.