Search

In this brief, low-frequency noise (LFN) characteristics are studied in oxide-based (\TiN/\HfOx/\Pt) resistive random access nemory cells having different dimensions. It is confirmed that, for the low resistance state (LRS), current conduction is localized without an area dependence, whereas for the high resistance state, it is a uniform leakage current throughout the whole device area. An LFN model is proposed for the filamentary LRS based on the carrier number fluctuation approach, allowing physical analysis of filament characteristics and the surrounding trap concentration. [ABSTRACT FROM AUTHOR]

Intensive systemic chemotherapy is the gold standard of acute myeloid leukemia (AML) treatment and is associated with considerable off-target toxicities. Safer and targeted delivery systems are thus urgently needed. In this study, we evaluated a virus-like particle derived from the human type 3 adenovirus, called the adenoviral dodecahedron (Dd) to target AML cells. The vectorization of leukemic cells was proved very effective at nanomolar concentrations in a time- and dose-dependent manner, without vector toxicity. The internalization involved clathrin-mediated energy-dependent endocytosis and strongly correlated with the expression of α V β 3 integrin. The treatment of healthy donor peripheral blood mononuclear cells showed a preferential targeting of monocytes compared to lymphocytes and granulocytes. Similarly, monocytes but also AML blasts were the best-vectorized populations in patients while acute lymphoid leukemia blasts were less efficiently targeted. Importantly, AML leukemic stem cells (LSCs) could be addressed. Finally, Dd reached peripheral monocytes and bone marrow hematopoietic stem and progenitor cells following intravenous injection in mice, without excessive spreading in other organs. These findings reveal Dd as a promising myeloid vector especially for therapeutic purposes in AML blasts, LSCs, and progenitor cells., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

Adenoviral dodecahedron is a virus-like particle composed of twelve penton base proteins, derived from the capsid of human adenovirus type 3. Due to the high cell penetration capacity, it was used as a vector for protein, peptide and drug delivery. Two receptors are known to be involved in the endocytic dodecahedron uptake, namely αv integrins and heparan sulfate proteoglycans. Since it has been observed, that dodecahedron efficiently penetrates a wide range of cancer cells, it suggests that other cellular compounds may play a role in the particle endocytosis. To shed some light onto the interactions with membrane lipids and their potential role in dodecahedron entry, we performed a series of experiments including biochemical assays, fluorescence confocal imaging of giant unilamellar vesicles and surface plasmon resonance, which indicated specific preference of the particle to anionic phosphatidylserine. Experiments performed on cholesterol-depleted epithelial cells showed that cholesterol is essential in the endocytic uptake, however a direct interaction was not observed. We believe that the results will allow to better understand the role of lipids in dodecahedron entry and to design more specific dodecahedron-based vectors for drug delivery to cancer cells., (Copyright © 2018. Published by Elsevier B.V.)

Virus-like particles (VLPs) assemble spontaneously during the viral cycle or in heterologous systems during expression of viral structural protein. Depending on the complexity of the VLPs, they can be obtained by expression in prokaryotic or eukaryotic expression system from the suitable recombinant vectors, or formed in cell-free conditions. Moreover, they can be built from proteins of a single virus, or can present the proteins or peptides derived from a virus or cell on a platform derived from any other single virus, thus forming chimeric VLPs. VLPs are best known for their immunogenic properties, but the versatility of VLPs allows a wide variety of applications. They are lately in the centre of investigations in vaccinology, drug delivery and gene therapy. This review focuses on utilization of VLPs for drug delivery.

This review presents data on commercial and experimental virus-like particle (VLP) vaccines, including description of VLP vaccines against influenza. Virus-like particles are multimeric, sometimes multiprotein nanostructures assembled from viral structural proteins and are devoid of any genetic material. VLPs present repetitive high-density displays of viral surface proteins. Importantly, they contain functional viral proteins responsible for cell penetration by the virus, ensuring efficient cell entry and thus tissue-specific targeting, determined by the origin of the virus. The foremost application of VLPs is in vaccinology, where they provide delivery systems that combine good safety profiles with strong immunogenicity and constitute a safe alternative to inactivated infectious viruses. These stable and versatile nanoparticles display excellent adjuvant properties capable of inducing innate and cognate immune responses. They present both, high-density B-cell epitopes, for antibody production and intracellular T-cell epitopes, thus inducing, respectively, potent humoral and cellular immune responses. Uptake of VLPs by antigen-presenting cells leads to efficient immune responses resulting in control of pathogenic microorganisms.

Background: The production process for the current influenza vaccine takes about 6 months and its antigenic composition must be modified annually. In the attempt towards developing influenza vaccine production that would be faster, safer and cheaper we engineered an influenza vaccine in which multiple copies of hemagglutinin (HA) would be delivered by a vector, adenovirus dodecahedron (Ad Dd). Dd is a virus-like particle, formed by assembly of twelve copies of pentameric penton base (Pb) proteins responsible for virus penetration. In order to attach HA to the vector, an adaptor containing WW domains was used. The WW domain is a linear peptide fragment identified as a partner of proline-proline-x-tyrosine (PPxY) motif present at the N-terminal extremity of the Pb protein, which is a building block of Dd. That tandem of three WW domains in fusion with the protein of interest enables interaction with Dd and efficient translocation to the cytoplasm of cells in culture., Results: Since HA is an oligomeric protein with complicated processing, we prepared six different constructs of HA (A/swan/Poland/467/2006(H5N1)) in fusion with the WW adaptor. Herein we report baculovirus expression and functional analysis of six HA-WW variants. The best behaving variant was successfully delivered into human cells in vitro., Conclusions: Engineering of a soluble complex of HA with Dd, a virus-like particle that serves as a vector, an adjuvant and as a multivalent presentation platform, is an important step toward a novel influenza vaccine.

Amitozyn (Am) is a semi-synthetic drug produced by the alkylation of major celandine (Chelidonium majus L.) alkaloids with the organophosphorous compound N,N'N'-triethylenethiophosphoramide (ThioTEPA). We show here that the treatment of living cells with Am reversibly perturbs the microtubule cytoskeleton, provoking a dose-dependent cell arrest in the M phase. Am changed the dynamics of tubulin polymerization in vitro, promoted the appearance of aberrant mitotic phenotypes in HeLa cells and induced apoptosis by the activation of caspase-9, caspase-3 and PARP, without inducing DNA breaks. Am treatment of HeLa cells induced changes in the phosphorylation of the growth suppressor pRb that coincided with maximum mitotic index. The dose-dependent and reversible anti-proliferative effect of Am was observed in several transformed cell lines. Importantly, the drug was also efficient against multidrug-resistant, paclitaxel-resistant or p53-deficient cells. Our results thus open the way to further pre-clinical evaluation of Am.

During the viral life cycle adenoviruses produce excess capsid proteins. Human adenovirus serotype 3 (Ad3) synthesizes predominantly an excess of free pentons, the complexes of pentameric penton base and trimeric fiber proteins, which are responsible for virus penetration. In infected cells Ad3 pentons spontaneously assemble into dodecahedral virus-like nano-particles containing twelve pentons. They also form in insect cells during expression in the baculovirus system. Similarly, in the absence of fiber protein dodecahedric particles built of 12 penton base pentamers can be produced. Both kinds of dodecahedra show remarkable efficiency of intracellular penetration and can be engineered to deliver several millions of foreign cargo molecules to a single target cell. For this reason, they are of great interest as a delivery vector. In order to successfully manipulate this potential vector for drug and/or gene delivery, an understanding of the molecular basis of vector assembly and integrity is critical. Crystallographic data in conjunction with site-directed mutagenesis and biochemical analysis provide a model for the molecular determinants of dodecamer particle assembly and the requirements for stability. The 3.8 Å crystal structure of Ad3 penton base dodecamer (Dd) shows that the dodecahedric structure is stabilized by strand-swapping between neighboring penton base molecules. Such N-terminal strand-swapping does not occur for Dd of Ad2, a serotype which does not form Dd under physiological conditions. This unique stabilization of the Ad3 dodecamer is controlled by residues 59-61 located at the site of strand switching, the residues involved in putative salt bridges between pentamers and by the disordered N-terminus (residues 1-47), as confirmed by site directed mutagenesis and biochemical analysis of mutant and wild type protein. We also provide evidence that the distal N-terminal residues are externally exposed and available for attaching cargo.

The majority of proteins are unable to translocate into the cell interior. Hence for peptide- and protein-based therapeutics a direct intracytoplasmic delivery with the aid of transducing agents is an attractive approach. We wanted to deliver to the cell interior a putatively cytotoxic protein VPg. Protein transduction was achieved in vitro with three different commercial products. However, in our hands, delivery of various control proteins without known deleterious effects, as well as of protein VPg, always induced cell death. Finally, we used a novel transducing peptide Wr-T, which was not toxic to cultured cells, even in a quite large range of concentrations. Most importantly, control protein delivered to cells in culture did not display any toxicity while VPg protein exerted a strong cytotoxic effect. These data show that results obtained with cell-penetrating agents should be interpreted with caution.

This review describes the properties of some rare eukaryotic chaperones that each assist in the folding of only one target protein. In particular, we describe (1) the tubulin cofactors, (2) p47, which assists in the folding of collagen, (3) α-hemoglobin stabilizing protein (AHSP), (4) the adenovirus L4-100 K protein, which is a chaperone of the major structural viral protein, hexon, and (5) HYPK, the huntingtin-interacting protein. These various-sized proteins (102-1,190 amino acids long) are all involved in the folding of oligomeric polypeptides but are otherwise functionally unique, as they each assist only one particular client. This raises a question regarding the biosynthetic cost of the high-level production of such chaperones. As the clients of faithful chaperones are all abundant proteins that are essential cellular or viral components, it is conceivable that this necessary metabolic expenditure withstood evolutionary pressure to minimize biosynthetic costs. Nevertheless, the complexity of the folding pathways in which these chaperones are involved results in error-prone processes. Several human disorders associated with these chaperones are discussed.

Virus-coded VPg protein of Potato virus Y (PVY) does not have homologs apart from other VPgs. Since VPg is indispensable for the potyvirus life cycle, it appeared a good candidate for eliciting pathogen-derived resistance to PVY. Following agroinfection used to obtain PVY VPg-transgenic Arabidopsis thaliana plants, only few transgenic seeds were recovered giving rise to six transgenic plants that contained the VPg gene with the correct sequence. They generated VPg mRNA, but VPg protein was not detected. Some plants were immune to PVY infection suggesting post-transcriptional gene silencing. However, the likely PVY VPg toxicity exerted at an early stage of transformed seeds development precludes its use for engineering pathogen-derived resistance.

Background: Bleomycin (BLM) is an anticancer antibiotic used in many cancer regimens. Its utility is limited by systemic toxicity and dose-dependent pneumonitis able to progress to lung fibrosis. The latter can affect up to nearly 50% of the total patient population, out of which 3% will die. We propose to improve BLM delivery by tethering it to an efficient delivery vector. Adenovirus (Ad) dodecahedron base (DB) is a particulate vector composed of 12 copies of a pentameric viral protein responsible for virus penetration. The vector efficiently penetrates the plasma membrane, is liberated in the cytoplasm and has a propensity to concentrate around the nucleus; up to 300000 particles can be observed in one cell in vitro., Principal Findings: Dodecahedron (Dd) structure is preserved at up to about 50 degrees C at pH 7-8 and during dialysis, freezing and drying in the speed-vac in the presence of 150 mM ammonium sulfate, as well as during lyophilization in the presence of cryoprotectants. The vector is also stable in human serum for 2 h at 37 degrees C. We prepared a Dd-BLM conjugate which upon penetration induced death of transformed cells. Similarly to free bleomycin, Dd-BLM caused dsDNA breaks. Significantly, effective cytotoxic concentration of BLM delivered with Dd was 100 times lower than that of free bleomycin., Conclusions/significance: Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs.

Potato virus Y (PVY) is a common potyvirus of agricultural importance, belonging to the picornavirus superfamily of RNA plus-stranded viruses. A covalently linked virus-encoded protein VPg required for virus infectivity is situated at the 5' end of potyvirus RNA. VPg seems to be involved in multiple interactions, both with other viral products and host proteins. VPgs of potyviruses have no known homologs, and there is no atomic structure available. To understand the molecular basis of VPg multifunctionality, we have analyzed structural features of VPg from PVY using structure prediction programs, functional assays, and biochemical and biophysical analyses. Structure predictions suggest that VPg exists in a natively unfolded conformation. In contrast with ordered proteins, PVY VPg is not denatured by elevated temperatures, has sedimentation values incompatible with a compact globular form, and shows a CD spectrum of a highly disordered protein, and HET-HETSOFAST NMR analysis suggests the presence of large unstructured regions. Although VPg has a propensity to form dimers, no functional differences were seen between the monomer and dimer. These data strongly suggest that the VPg of PVY should be classified among intrinsically disordered proteins. Intrinsic disorder lies at the basis of VPg multifunctionality, which is necessary for virus survival in the host.

Exogenous proteinase inhibitors are valuable and economically interesting protective biotechnological tools. We examined whether small proteinase inhibitors when fused to a selected target protein can protect the target from proteolytic degradation without simultaneously affecting the function and activity of the target domain. Two proteinase inhibitors were studied: a Kazal-type silk proteinase inhibitor (SPI2) from Galleria mellonella, and the Cucurbita maxima trypsin inhibitor I (CMTI I). Both inhibitors target serine proteinases, are small proteins with a compact structure stabilized by a network of disulfide bridges, and are expressed as free polypeptides in their natural surroundings. Four constructs were prepared: the gene for either of the inhibitors was ligated to the 5' end of the DNA encoding one or the other of two selected target proteins, the coat protein (CP) of Potato potyvirus Y or the Escherichia coli beta-glucuronidase (GUS). CMTI I fused to the target proteins strongly hampered their functions. Moreover, the inhibitory activity of CMTI I was retained only when it was fused to the CP. In contrast, when fused to SPI2, specific features and functions of both target proteins were retained and the inhibitory activity of SPI2 was fully preserved. Measuring proteolysis in the presence or absence of either inhibitor, we demonstrated that proteinase inhibitors can protect target proteins used either free or as a fusion domain. Interestingly, their inhibitory efficiency was superior to that of a commercial inhibitor of serine proteinases, AEBSF.

The use of cytotoxic agents to eliminate cancer cells is limited because of their nonselective toxicity and unwanted side effects. One of the strategies to overcome these limitations is to use latent prodrugs that become toxic in situ after being enzymatically activated in target cells. In this work we describe a method for producing tumor-specific toxins by using a ubiquitin fusion technique. The method is illustrated by the production of recombinant toxins by in-frame fusion of ubiquitin to saporin, a toxin from the plant Saponaria officinalis. Ubiquitin-fused toxins were rapidly degraded via the ubiquitin-proteasome system, significantly reducing their nonspecific toxicity. The insertion of the protease-cleavage sequence between ubiquitin and saporin led to the removal of ubiquitin by the protease and resulted in protease-dependent stabilization of the toxin. We engineered toxins that can be stabilized by specific proteases such as deubiquitinating enzymes and prostate-specific antigen (PSA). Both constructs were activated in vitro and in cultured cells by the appropriate enzyme. Processing by the protease resulted in a greater than 10-fold increase in the toxicity of these constructs. Importantly, the PSA-cleavable toxin was able to kill specifically the PSA-producing prostate cancer cells. The ubiquitin fusion technique is thus a versatile and reliable method for obtaining selective cytotoxic agents and can easily be adapted for different kinds of toxins and activating proteases.

The adenovirus penton, a noncovalent complex of the pentameric penton base and trimeric fiber proteins, comprises the vertices of the adenovirus capsid and contains all necessary components for viral attachment and internalization. The 3.3 A resolution crystal structure of human adenovirus 2 (hAd2) penton base shows that the monomer has a basal jellyroll domain and a distal irregular domain formed by two long insertions, a similar topology to the adenovirus hexon. The Arg-Gly-Asp (RGD) motif, required for interactions with cellular integrins, occurs on a flexible surface loop. The complex of penton base with bound N-terminal fiber peptide, determined at 3.5 A resolution, shows that the universal fiber motif FNPVYPY binds at the interface of adjacent penton base monomers and results in a localized structural rearrangement in the insertion domain of the penton base. These results give insight into the structure and assembly of the adenovirus capsid and will be of use for gene-therapy applications.

Ubiquitylation, the modification of cellular proteins by the covalent attachment of ubiquitin, is critical for diverse biological processes including cell cycle progression, signal transduction and stress response. This process can be reversed and regulated by a group of proteases called deubiquitylating enzymes (DUBs). Otubains are a recently identified family of DUBs that belong to the ovarian tumour (OTU) superfamily of proteins. Here, we report the first crystal structure of an OTU superfamily protein, otubain 2, at 2.1 A resolution and propose a model for otubain-ubiquitin binding on the basis of other DUB structures. Although otubain 2 is a member of the cysteine protease superfamily of folds, its crystal structure shows a novel fold for DUBs. Moreover, the active-site cleft is sterically occluded by a novel loop conformation resulting in an oxyanion hole, which consists uniquely of backbone amides, rather than the composite backbone/side-chain substructures seen in other DUBs and cysteine proteases. Furthermore, the residues that orient and stabilize the active-site histidine of otubain 2 are different from other cysteine proteases. This reorganization of the active-site topology provides a possible explanation for the low turnover and substrate specificity of the otubains.

Poneratoxin is a small neuropeptide found in the venom of the ant Paraponera clavata. It is stored in the venom reservoir as an inactive 25-residue peptide. Here we describe both chemically synthesized poneratoxin and poneratoxin obtained by expression in insect cells. When expressed in insect cells, poneratoxin was observed attached to cell membranes. Both synthetic and recombinant ponerotoxins were soluble below pH 4.5. The structure of synthetic poneratoxin was characterized by circular dichroism and solved by nuclear magnetic resonance. In an environment imitating a lipid bilayer, at pH within the range of insect hemolymph, synthetic poneratoxin has a V shape, with two alpha-helices connected by a beta-turn. Insect larvae were paralyzed by injection of either of the purified toxins, with the recombinant one acting faster. The recombinant toxin-producing baculovirus reduced the average survival time of the insect host by 25 h compared with unmodified virus. Mass spectrometry analysis showed that the recombinant toxin has an N-terminal 21-residue extension, possibly improving its stability and/or stabilizing the membrane-bound state. The potential use of poneratoxin for the construction of biological insecticide is discussed.

Human enteric adenovirus Ad41 is associated with children gastroenteritis. To infect gastrointestinal cells, the invading virus must be acid-stable and resistant to inactivation by bile salts and proteases. In addition, it has to cross the mucus barrier before it infects mucosa cells. We show that Ad41 infectivity is not diminished by acid exposure, a condition limiting the infectivity of the respiratory Ad. This feature can be attributed to a large extent to the global basic charge of enteric Ad virions and to the stability of Ad41 fiber, a viral protein mediating virus attachment. Upon exposure to pH shock, the respiratory Ad2 loses its ability to interact with lipids while enteric Ad41 still binds to the major phospholipids of gastric and intestine mucus. In addition, contrary to respiratory Ad, enteric Ad41 interacts with several sphingolipid components of plasma membranes. These results show that the molecular bases of the Ad41 enteric tropism stem from its particular physicochemical properties.

During adenovirus type 3 (Ad3) infection cycle, the penton (Pt) of the viral capsid, a noncovalent complex of fiber and penton base proteins, is produced in large excess and self-assembles to form a highly organized dodecahedral structure, termed dodecahedron (Dd). The physiological role of these particles is poorly understood, but we have recently reported that they can penetrate cells with high efficiency and thus may constitute an attractive tool for gene or protein delivery approaches. Surprisingly, Dd displayed the ability to enter cells non-permissive to Ad3, suggesting the existence of additional internalization modes. In this study, we show that Ad3 Dd binds to cell surface heparan sulfate (HS) through high affinity interaction with the penton base. Furthermore, binding to HS was found to be the prerequisite for a novel and Dd specific entry pathway that could not be used by Ad3. Overall, these data provide new insights in the possible role of Dd during viral infection and potential therapeutic applications.

The modification of cellular proteins by ubiquitin (Ub) is an important event that underlies protein stability and function in eukaryotes. Protein ubiquitylation is a dynamic and reversible process; attached Ub can be removed by deubiquitylating enzymes (DUBs), a heterogeneous group of cysteine proteases that cleave proteins precisely at the Ub-protein bond. Two families of DUBs have been identified previously. Here, we describe new, highly specific Ub iso-peptidases, that have no sequence homology to known DUBs, but which belong to the OTU (ovarian tumour) superfamily of proteins. Two novel proteins were isolated from HeLa cells by affinity purification using the DUB-specific inhibitor, Ub aldehyde (Ubal). We have named these proteins otubain 1 and otubain 2, for OTU-domain Ubal-binding protein. Functional analysis of otubains shows that the OTU domain contains an active cysteine protease site.

Adenovirus (Ad) is an airborne, nonenveloped virus infecting respiratory epithelium. To study the mechanism of Ad entry, we used alveolar adenocarcinoma A549 cells, which have retained the ability of alveolar epithelial type II cells to synthesize the major component of pulmonary surfactant, disaturated phosphatidylcholine. Stimulation of phosphatidylcholine secretion by calcium ionophore or phorbol ester augmented the susceptibility of these cells to Ad. Both Ad infection and recombinant-Ad-mediated transfection increased in the presence of dipalmitoyl phosphatidylcholine (DPPC) liposomes in culture medium. Importantly, in the presence of DPPC liposomes, virus penetrates the cells independently of virus-specific protein receptors. DPPC vesicles bind Ad and are efficiently incorporated by A549 lung cells, serving as a virus vehicle during Ad penetration. To identify the viral protein(s) mediating Ad binding, a flotation of liposomes preincubated with structural viral proteins was employed, showing that the only Ad protein bound to DPPC vesicles was a hexon. The hexon preserved its phospholipid-binding properties upon purification, confirming its involvement in virus binding to the phospholipid. Given that disaturated phosphatidylcholine not only covers the inner surface of alveoli in the lungs but also reenters alveolar epithelium during lung surfactant turnover, Ad binding to this phospholipid may provide a pathway for virus entry into alveolar epithelium in vivo.

The invasion strategy of many viruses involves the synthesis of viral gene products that mimic the functions of the cellular proteins and thus interfere with the key cellular processes. Here we show that adenovirus infection is accompanied by an increased ubiquitin-cleaving (deubiquitinating) activity in the host cells. Affinity chromatography on ubiquitin aldehyde (Ubal), which was designed to identify the deubiquitinating proteases, revealed the presence of adenovirus L3 23K proteinase (Avp) in the eluate from adenovirus-infected cells. This proteinase is known to be necessary for the processing of viral precursor proteins during virion maturation. We show here that in vivo Avp deubiquitinates a number of cellular proteins. Analysis of the substrate specificity of Avp in vitro demonstrated that the protein deubiquitination by this enzyme could be as efficient as proteolytic processing of viral proteins. The structural model of the Ubal-Avp interaction revealed some similarity between S1-S4 substrate binding sites of Avp and ubiquitin hydrolases. These results may reflect the acquisition of an advantageous property by adenovirus and may indicate the importance of ubiquitin pathways in viral infection.

Enteric adenoviruses of serotypes 40 and 41 possess some specific structural features, one of which is the presence on the virion of two fibers of different lengths and primary sequences. These viruses are notoriously difficult to grow under laboratory conditions. In this paper the successful growth and purification of Ad41 are presented in detail. Structural Ad41 proteins were analyzed by biochemical methods, mass spectrometry, and electron microscopy (EM), in order to identify and localize them on polyacrylamide denaturing gels and to assess the proportion of short and long fibers in the virion. Surprisingly, the three proteins composing virus short and long pentons did not totally enter the denaturing polyacrylamide gels, which is probably due in part to their high pI. The pentons were separately purified and their dimensions were estimated from EM data. The EM images suggest that there are the same amounts of short and long fibers in each virion.

Adenoviral gene therapy vectors suffer from the disadvantages of toxicity and immunogenicity associated with the expression of adenoviral genes from the vector backbone. We report here an alternative strategy for gene delivery that utilizes a single component of the adenoviral type 7 capsid, the penton base (Ad7PB). The Ad7PB gene was sequenced and its amino-acid composition was deduced from its nucleotide sequence. The penton was expressed in Escherichia coli as a soluble C-terminal fusion with glutathione S-transferase (GST-Ad7PB) and was purified by single-step affinity chromatography. Both GST-Ad7PB and cleaved (GST-free) Ad7PB retained the ability to fold into pentamers as observed by electron microscopy. GST-Ad7PB was able to bind a synthetic peptide (FK20) derived from the Ad type 7 fiber and retard DNA through a polylysine chain present at the C-terminus of this linker peptide. GST-Ad7PB was an effective cell transfecting agent when assayed on 293 cells. Transfection was not dependent upon the presence of lysosomotropic agents indicating efficient endosome escape capability. Excess of an RGD-containing peptide derived from Ad7PB was able to inhibit transfection indicating specific integrin-mediated uptake of the GST-Ad7PB-FK20-DNA complexes. We propose that Ad7 pentons can be developed into integrin-specific gene delivery agents.

Background: Intracellular release of free DNA from the vector complex is one of the critical steps limiting the efficiency of non-viral gene delivery. The complex should be stable enough to prevent DNA degradation but it should be destabilized inside the cell to allow DNA release and transcription. Destabilization and degradation of synthetic vectors is also required to reduce their cytotoxicity and augment the life-time of transfected cells., Results: Here we describe new cationic amphiphiles made from the natural pro-vitamin, lipoic acid, that reversibly binds and releases DNA, depending on the redox state of the lipoate moieties. In the oxidized state these amphiphiles condense DNA into homogeneous spherical particles, which, upon reduction, swell into DNA toroids with subsequent release of free DNA. Complex reduction and DNA release can be induced by various thiols as well as enzymatically, by thioredoxin reductase. Transfection with amphiphile-DNA complexes in vitro shows a several fold increase of transgene expression compared with DOTAP, and can be further augmented by attachment of the nucleus-targeting peptide to the amphiphile. The increase of transfection efficiency results from GSH- and NAD(P)H-dependent complex reduction and release of free DNA inside the cells., Conclusions: The present work demonstrates the principle of a redox-controlled gene delivery system that uses the reversibility of thiol-disulfide exchange reaction. Our data suggest that the efficiency of synthetic vectors can be augmented by their controlled destabilization inside the cells. Being formed from the natural non-toxic compound lipoic acid, these cationic amphiphiles provide a new promising class of synthetic vectors for gene delivery.