Efficient intracellular delivery of a protein and a low molecular weight substance via recombinant polyomavirus-like particles

Efficient encapsulation of foreign molecules like proteins and low molecular weight drugs into polyoma virus-like particles (capsoids) was achieved by the development of an anchoring technique based upon the specific interaction of the inner core protein VP2 with VP1 pentamers. A stretch of 49 amino acids of VP2 served as an anchor molecule, either expressed as a fusion protein with green fluorescent protein (GFP) or covalently linked to methotrexate (MTX). The loaded capsoids showed regular morphology and stability for several months. GFP and MTX were internalized into cells in vitro, as was demonstrated by the detection of GFP and VP1 fluorescence in mouse fibroblasts and the cytostatic effect of intracellularly released MTX on leukemia T cells.     

Site-specific fluorescence labelling of recombinant polyomavirus-like particles

For the development of gene therapy protocols based on polyomavirus-like particles, we describe a method for fluorescence labelling of virions in order to study virus-cell interactions preceding gene delivery. Site-specific fluorescence labelling of polyomavirus-like particles is achieved via a single cysteine residue and maleimide conjugates of fluorescence dyes (fluorescein, Texas Red). Polyomavirus-like particles can be assembled in vitro from recombinant capsomers produced in E. coli. Since the assembly process is independent of disulfide bond formation, all cysteine residues of the wild-type protein were replaced by serines, and a new unique cysteine residue was introduced for the attachment of the fluorescence marker.     

Ganglioside-dependent cell attachment and endocytosis of murine polyomavirus-like particles

For murine polyomavirus (Py), previous studies suggest the cellular target is a terminal alpha2,3-linked sialic acid. Here, we investigate the binding and uptake of mouse polyomavirus-like particles (PyVLP) derived from bacterially expressed VP1. We find that in fibroblast 3T6 cells, binding of PyVLP was substantially reduced by sialidase treatment, but only moderately affected by protease treatment, suggesting glycolipids such as the sialic acid-containing gangliosides mediate cell attachment. We further tested the entry requirement of PyVLP using the ganglioside-deficient GM95 murine cell line, and find PyVLP binding and entry were reduced in these cells. Finally, we find that addition of gangliosides G(M1), G(D1a), and G(T1b) to GM95 cells restored cellular PyVLP binding and uptake. Taken together, results indicate that gangliosides function in PyVLP cell attachment and endocytosis.     

Mechanism of assembly of recombinant murine polyomavirus-like particles

VP1 is the major viral coat protein of murine polyomavirus and can be used for the generation of virus-like particles in vitro. Here, we demonstrate that capsid assembly is an equilibrium reaction followed by oxidation of intracapsomere disulfide bonds, which are not essential for the formation of virus-like particles but enable complete particle assembly and prevent capsid disassembly.     

T-helper cell-mediated proliferation and cytokine responses against recombinant Merkel cell polyomavirus-like particles

The newly discovered Merkel Cell Polyomavirus (MCPyV) resides in approximately 80% of Merkel cell carcinomas (MCC). Causal role of MCPyV for this rare and aggressive skin cancer is suggested by monoclonal integration and truncation of large T (LT) viral antigen in MCC cells. The mutated MCPyV has recently been found in highly purified leukemic cells from patients with chronic lymphocytic leukemia (CLL), suggesting a pathogenic role also in CLL. About 50-80% of adults display MCPyV-specific antibodies. The humoral immunity does not protect against the development of MCC, as neutralizing MCPyV antibodies occur in higher levels among MCC patients than healthy controls. Impaired T-cell immunity has been linked with aggressive MCC behavior. Therefore, cellular immunity appears to be important in MCPyV infection surveillance. In order to elucidate the role of MCPyV-specific Th-cell immunity, peripheral blood mononuclear cells (PBMC) of healthy adults were stimulated with MCPyV VP1 virus-like particles (VLPs), using human bocavirus (HBoV) VLPs and Candida albicans antigen as positive controls. Proliferation, IFN-γ, IL-13 and IL-10 responses were examined in 15 MCPyV-seropositive and 15 seronegative volunteers. With the MCPyV antigen, significantly stronger Th-cell responses were found in MCPyV-seropositive than MCPyV-seronegative subjects, whereas with the control antigens, the responses were statistically similar. The most readily detectable cytokine was IFN-γ. The MCPyV antigen tended to induce stronger IFN-γ responses than HBoV VLP antigen. Taken together, MCPyV-specific Th-cells elicit vigorous IFN-γ responses. IFN-γ being a cytokine with major antiviral and tumor suppressing functions, Th-cells are suggested to be important mediators of MCPyV-specific immune surveillance.     

Yeast cells allow high-level expression and formation of polyomavirus-like particles

Polyomavirus-derived virus-like particles (VLPs) have been described as potential carriers for encapsidation of nucleic acids in gene therapy. Although VLPs can be generated in E. coli or insect cells, the yeast expression system should be advantageous as it is well established for the biotechnological generation of products for human use, especially because they are free of toxins hazardous for humans. We selected the yeast Saccharomyces cerevisiae for expression of the major capsid protein VP1 of a non-human polyomavirus, the hamster polyomavirus (HaPV). Two entire HaPV VP1-coding sequences, starting with the authentic and a second upstream ATG, respectively, were subcloned and expressed to high levels in Saccharomyces cerevisiae. The expressed VP1 assembled spontaneously into VLPs with a structure resembling that of the native HaPV capsid. Determination of the subcellular localization revealed a nuclear localization of some particles formed by the N-terminally extended VP1, whereas particles formed by the authentic VP1 were found mainly in the cytoplasmic compartment.     

Protein delivery by the cell-penetrating peptide YTA2

In most cases, the transport of cell-penetrating peptide (CPP) with a cargo molecule over the plasma membrane requires a cross-linking of the cargo molecule to the peptide. Lately, a method of cargo delivery, coincubation with CPP, has been applied. We have studied uptake and toxicity of the CPP, YTA2, in the Bowes human melanoma cell line and human MDA-MB-231 breast cancer cell line and compared the results with known cell-penetrating peptides. The results show that fluoresceinyl YTA2 is taken up by the Bowes cells with 3.23 nmol/mg protein and shows low membrane toxicity to the cells with an EC50 of 60 microM. Furthermore, we show that YTA2 is capable of delivering cargo proteins, such as beta-galactosidase and tetramethyl rhodamine iso-thiocyanate (TRITC) labeled streptavidin into cells by coincubation. The delivery of TRITC-labeled streptavidin was quantified to 42.4 pmol streptavidin/mg protein. The delivery of proteins into the cells by mere coincubation is an advantage, since the chemical coupling between the CPP and the cargo molecule, which adds time-consuming synthesis and purification steps, can be omitted. In addition, the flexibility in CPP cargo delivery is increased.     

Protein transduction domain peptide mediates delivery to the brain via the blood-brain barrier in Drosophila melanogaster

The phenomenon of protein transduction represents internalization of short peptides known as protein transduction domains (PTD) by cells. It is widely used in the development of new preparations for treatment of various brain disorders. However, the drug discovery process is limited by lack of simple and reliable models of blood brain barrier (BBB). These models should meet two main criteria: they should be applicable for testing of large numbers of samples simultaneously reproduce the physiological and functional characteristics of mammalian (including) human BBB. The major goal of this study was to estimate the BBB-crossing ability of known PTD-peptides using Drosophila melanogaster BBB as the model. We demonstrate here that after abdominal administration the PTD-peptide penetratin, derived from a Drosophila Antennapedia homeodomain protein can cross Drosophila and deliver the apoE mimetic peptide exhibiting neuroprotective properties.     

Tumor-targeted gene delivery using molecularly engineered hybrid polymers functionalized with a tumor-homing peptide

Before gene therapy can be used in clinical settings, safe and efficient DNA delivery systems must be developed to overcome a range of extra- and intracellular transport barriers. As a step toward the development of a modular, multifunctional gene delivery system to overcome these diverse barriers, we have developed a family of linear-dendritic "hybrid" polymers which contain functionalities for tissue targeting, minimization of nonspecific interactions, endosomal buffering, and DNA binding. Here, we demonstrate the rapid three-step, room-temperature, aqueous synthesis of hybrid polymers, as well as the functionalization of these polymers with a peptide targeting ligand that specifically binds to glucose-regulated protein-78 kDa (GRP-78), a clinically relevant tumor antigen identified in human cancer patients. These polymer systems can condense plasmid DNA into small nanoparticle structures (<210 nm) and transfect cells expressing GRP-78 with efficiencies that exceed that of branched polyethylenimine (bPEI), one of the best commercially available polymers for in vitro transfections. The synthetic approach described here may be useful for the rapid synthesis and optimization of polymer gene delivery systems bearing a range of diverse functional domains, and the specific GRP-78-targeted systems developed in this study may potentially have clinical applications in cancer gene therapy.     

Intracellular delivery of nanometric DNA particles via the folate receptor

The size of condensed DNA particles is a key determinant for both diffusion to target cells in vivo and intracellular trafficking. The smallest complexes are obtained when each DNA molecule collapses individually. This was achieved using a designed cationic thiol-detergent, tetradecyl-cysteinyl-ornithine (C(14)COrn). The resulting particles were subsequently stabilized by air-induced dimerization of the detergent into a disulfide lipid on the DNA template. Particles are anionic (zeta potential = -45 mV), and their size (30 nm) corresponds to the volume of a single plasmid DNA molecule. The electrophoretic mobility of the condensed DNA, though quasi-neutralized, was found higher than that of the extended DNA. Moreover, the dimerized (C(14)COrn)(2) lipid was found to be an efficient transfection reagent for various cell lines. In an attempt to achieve extended circulation times and to target tumors by systemic delivery, we have coated the particles with PEG-folate residues. Plasmid DNA was condensed into monomolecular particles as described above and coated by simple mixing with DPPE-PEG-folate. Physicochemical measurements showed particles coated with 2% of DPPE-PEG(3400)-folate remain monomolecular and are stable in the cell-culture medium. Caveolae-mediated cell entry was demonstrated by ligand-dependence, by competition with excess folic acid as well as by confocal microscopy.     

Transdermal protein delivery by a coadministered peptide identified via phage display

Efficient transdermal drug delivery of large hydrophilic drugs is challenging. Here we report that the short synthetic peptide, ACSSSPSKHCG, identified by in vivo phage display, facilitated efficient transdermal protein drug delivery through intact skin. Coadministration of the peptide and insulin to the abdominal skin of diabetic rats resulted in elevated systemic levels of insulin and suppressed serum glucose levels for at least 11 h. Significant systemic bioavailability of human growth hormone was also achieved when topically coadministered with the peptide. The transdermal-enhancing activity of the peptide was sequence specific and dose dependent, did not involve direct interaction with insulin and enabled penetration of insulin into hair follicles beyond a depth of 600 microm. Time-lapse studies suggested that the peptide creates a transient opening in the skin barrier to enable macromolecular drugs to reach systemic circulation.     

Transdermal peptide delivery

The transdermal delivery of peptide drugs, though ill-favoured by their hydrophilicity and high molecular mass, would seem very attractive from the pharmacotherapeutical and patient compliance point of view. In some cases, effective transdermal dosing has been achieved in vivo, especially with the aid of iontophoresis. This paper deals with a dodecapeptide, des-enkephalin-gamma-endorphin, of which the transepidermal permeation and the intra(epi-)dermal biotransformation were both studied in vitro. Small, though measurable, fluxes through human stratum corneum were obtained in vitro, which could be enhanced by using a skin lipid fluidizer. The half-life of the peptide, both in the epidermis and in the dermis, was surprisingly long as compared with that in human plasma. Hence, improvement of the transdermal bioavailability of the peptide will most likely be obtained chiefly by enhancing its flux (possibly through iontophoresis), intra(epi-)dermal degradation being a problem of only minor importance.     

基因工程疫苗的病毒载体

病毒基因工程疫苗是以活病毒为载体将一段外源基因导入机体细胞内,并使外源基因维持较高水平的表达。通过使用复制型或复制缺陷型载体能使表达的抗原诱生机体产生相应的体液抗体,并能引起机体产生细胞介导的免疫反应及粘膜免疫反应。本文主要介绍有可能用于基因工程疫苗的DNA及RNA病毒载体构建及其应用。     

Formation of polyomavirus-like particles with different VP1 molecules that bind the urokinase plasminogen activator receptor

Icosahedral virus-like particles formed by the self-assembly of polyomavirus capsid proteins (Py-VLPs) can serve as useful nanostructures for delivering nucleic acids, proteins, and pharmaceuticals into animal cells and tissues. Four predominant surface-exposed loops in the VP1 structure offer potential sites to display sequences that might contribute new targeting specificities. Introduction into each of these loops of sequences derived from the amino-terminal fragment of urokinase plasminogen activator (uPA) or a related phage display peptide reduced the solubility of VP1 molecules when expressed in insect cells, and insertions into the EF loop reduced VP1 solubility least. Coexpression in insect cells of the uPA-VP1 molecules and VP1 containing a FLAG epitope in the HI loop permitted the formation of heterotypic Py-VLPs containing uPA-VP1 and FLAG-VP1. These heterotypic VLPs bound to uPAR on the surfaces of animal cells. Heterotypic Py-VLPs containing ligands for multiple cell surface receptors should be useful for targeting specific cells and tissues.     

Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells

The protein delivery across cellular membranes or compartments is limited by low biomembrane permeability because of the hydrophobic characteristics of cell membranes. Usually the delivery processes utilize passive protein channels or active transporters to overcome the membrane impediment. In this report, we demonstrate that arginine-rich intracellular delivery (AID) peptide is capable of efficiently delivering fused fluorescent proteins unpreferentially into different plant tissues of both tomato (a dicot plant) and onion (a monocot plant) in a fully bioactive form. Thus, cellular internalization via AID peptide can be a powerful tool characterized by its simplicity, non-invasion and high efficiency to express those bioactive proteins in planta or in plant cells in vivo. This novel method may alternatively provide broader applications of AID chimera in plants without the time-consuming transgenic approaches.     

Receptor-mediated delivery of engineered nucleases for genome modification

Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures.