A sustained passion for intracellular trafficking

I am honored to be the first recipient of the Women in Cell Biology Sustained Excellence in Research Award. Since my graduate school days, I have enjoyed being part of a stimulating scientific community the American Society for Cell Biology embodies. Having found myself largely by accident in a career that I find deeply enjoyable and fulfilling, I hope here to convey a sense that one need not have a “grand plan” to have a successful life in science. Simply following one''s interests and passions can sustain a career, even though it may involve some migration.     

Targeted adenoviral vectors

Replication-defective vectors based on human adenovirus serotypes 2 and 5 (Ad2 and Ad5) possess a number of attributes which favor their use as gene delivery vehicles in gene therapy applications. However, the widespread distribution of the primary cellular receptor for Ad, the coxsackievirus and adenovirus receptor (CAR), allows Ad vectors to infect a broad range of cells in the host. Conversely, a number of tissues which represent important targets for gene therapy, such as the airway epithelium and cancer cells, are refractory to Ad infection due a paucity of CAR. Thus, there is a strong rationale for the development of CAR-independent Ad vectors capable of enhanced specificity and efficiency of gene transfer to target cells. In this article we review the approaches which have been employed to generate tropism-modified Ad vectors. These targeting strategies have led to improvements in the safety and efficacy of Ad vectors and have the potential to yield an increased therapeutic benefit in the human clinical context.     

Construction of adenoviral vectors

Recombinant adenovirus vectors have proven to be useful tools in facilitating gene transfer. Construction of such vectors requires a knowledge of the adenovirus genome structure and its life cycle. A commonly used recombinant adenovirus involves deletion of the E1 region; such a recombinant is traditionally produced by overlap recombination after contransfection of 293 cells with a plasmid shuttle vector and a large right-end restriction fragment of viral DNA. The shuttle vector contains a cassette for a transgene placed in region E1 and flanking sequences from adenovirus for recombination. Normally, a high background of parental virus results because of the difficulty in separating right-end restriction fragment length DNA from uncut DNA. This paper describes a negative selection based on the traditional cotransfection method using viral DNA from an E1-deleted adenoviral recombinant that expresses green fluorescent protein (GFP). In situ fluorescent microscopy is used to distinguish the recombinant plaques (white or nonfluorescent) from the parental virus plaques (green or fluorescent). In addition, this system allows for the detection of contaminating parental virus at later stages when production lots of the recombinant vector are being made.     

Impaired intracellular trafficking of adeno-associated virus type 2 vectors limits efficient transduction of murine fibroblasts

Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful alternative to the more commonly used retrovirus- and adenovirus-based vectors for human gene therapy, efficient gene transfer and transgene expression by AAV vectors require that the following two obstacles be overcome. First, the target cell must express the receptor and the coreceptor for AAV infection, and second, the cell must allow for viral second-strand DNA synthesis. We now describe a third obstacle, impaired intracellular trafficking of AAV to the nucleus, which results in the lack of transgene expression in murine fibroblasts which do express the AAV receptor and the coreceptor and which are permissive for viral second-strand DNA synthesis. We document that AAV vectors bind efficiently and gain entry successfully into NIH 3T3 cells, but trafficking into the nucleus is significantly impaired in these cells. In contrast, viral trafficking to the nucleus in cells known to be efficiently transduced by AAV vectors is both rapid and efficient. The demonstration of yet another obstacle in AAV-mediated gene transfer has implications for the optimal use of these vectors in human gene therapy.     

Evaluation of adenoviral vectors by flow cytometry

Biological assays for adenoviral gene therapy vectors have included conventional procedures initially developed to detect wild-type adenoviruses. Standard virological assays to quantitate adenoviruses rely on the virus to infect and replicate in the host cell until a cytopathic effect is observed. The appearance of plaques, colonies of rounded, enlarged cells containing infectious virions, usually takes 2 to 3 weeks to reach an endpoint. We describe a flow cytometric bioassay for adenovirus which shortens the time from when the infection takes place to the time that biological titer is determined. A fluorescent focus-forming assay was one of the first rapid adenoviral bioassays developed. Virus titer was determined using fluorescence immunocytochemistry to detect adenovirus proteins and microscopy to count fluorescent foci in cultures of adenovirus-infected cells. In this study, we describe a flow cytometric assay performed on cells stained for adenovirus hexon capsid protein, where virus titer is determined based on the dose-dependent appearance of hexon-positive cells. Adenovirus hexon detection in infected cells can provide data to determine virus titer, inducible promoter function in vector-complementing cells, and vector replication in complementation-deficient cells.     

腺病毒载体在生物治疗中的应用

腺病毒载体是目前重要的基因转移载体之一。腺病毒可作为真核基因表达载体,可制成灭活、重组或抗癌疫苗用于预防呼吸道疾病、癌症和肝炎等传染病。在癌症的基因治疗方面,Ａｄ 载体可运载肿瘤抑制基因,自身基因编码蛋白能诱导细胞调亡,可作为前药物感染细胞,还能利用Ａｄ 的一些特殊复制子,达到治疗肿瘤的目的     

Production of adenoviral vectors and its recovery

The current demands for adenoviral vectors are increasing to satisfy pre-clinical and clinical gene therapy protocols. Consequently, there is a necessity of methodologies to improve production and recovery of intact particles with the minimum effect upon bioactivity. The production of adenoviral vectors in HEK 293 cells and the potential of an alternative aqueous two-phase system (ATPS) composed of PEG 300-phosphate in recovery of adenoviral vectors were investigated. The production of adenoviral vectors was carried out using a 2 L bioreactor equipped with two Rushton impellers. Different parameters including initial cell density, harvesting time and the addition of a buffer (HEPES) were studied in order to improve the production of adenoviral vectors in HEK 293 cells. A yield of 8 × 10¹¹ infective particles was achieved under the conditions characterized by the addition of Pluronic F-68, inoculation at an initial cell density of 3.5 × 10⁵ cells/mL and harvest of infected cells at 48 h post infection (hpi). This material was used for the evaluation of the ATPS recovery processes. It was demonstrated that the chemical components of the ATPS did not have a significant effect upon the infectivity of the adenoviral vectors and a total recovery of approximately 90% was obtained. These findings contribute to the process development for the manufacture of adenoviral vectors and other nanoparticulate bioproducts.     

A novel approach using transcomplementing adenoviral vectors for gene therapy of adrenocortical cancer.

Current therapies for adrenocortical carcinomas do not improve the life expectancy of patients. In this study, we tested whether a gene-transfer therapy based upon a suicide gene/prodrug system would be effective in an animal model of the disease. We employed E4- and E1A/B-depleted, herpes simplex virus-thymidine kinase-expressing adenoviral mutants that transcomplement each other within tumor cells, hereby improving transgene delivery and efficacy by viral replication in situ. Transcomplementation of vectors increased the fraction of transduced of tumor cells. This increase was accompanied by greater tumor volume reduction compared to non-transcomplementing approaches. Survival time improved with non-replicating vectors plus GCV compared to controls. However, transcomplementation/replication of vectors led to a further significant increment in anti-tumor activity and survival time (p < 0.02). In treated animals, we observed a high number of apoptotic nuclei both adjacent to and distant from injection sites and sites of viral oncolysis. Ultrastructural analyses exhibited nuclear inclusion bodies characteristic of virus production in situ, and provided further evidence that this therapy induced apoptotic cell death within tumor cells. We conclude that the efficacy of suicide gene therapy is significantly amplified by viral replication and, in combination with GCV, significantly reduces tumor burden and increases survival time.     

A system for the propagation of adenoviral vectors with genetically modified receptor specificities.

The development of genetically modified adenovirus (Ad) vectors with specificity for a single cell type will require both the introduction of novel tropism determinants and the ablation of endogenous tropism. Consequently, it will not be possible to exploit the native cellular entry pathway in the propagation of these targeted Ad vectors. Based on the concept that Ad enters cells by a two-step process in which a primary receptor serves as a high affinity binding site for the Ad fiber knob, with subsequent internalization mediated by alpha v integrins, we designed two artificial primary receptors. The extracellular domain of one of these synthetic receptors was derived from a single-chain antibody (sFv) with specificity for Ad5 knob, while the second receptor consisted of an icosapeptide identified by biopanning a phage display library against Ad5 knob. Expression of either of these artificial virus-binding receptors in fiber receptor-negative cells possessing alpha v integrins conferred susceptibility to Ad infection. We then created a novel mechanism for cell binding by genetically modifying both the vector and the target cell. In this approach, six histidine (His) residues were incorporated at the C-terminal of the Ad fiber protein. The resultant Ad vector was able to infect nonpermissive cells displaying the cognate artificial receptor, containing an anti-His sFv. This strategy, comprising a genetically engineered Ad virion and a modified cell line, should be useful in the propagation of targeted Ad vectors that lack the ability to bind the native fiber receptor.     

Using inducible vectors to study intracellular trafficking of GFP-tagged steroid/nuclear receptors in living cells

Intracellular trafficking and localization of proteins can now be efficiently visualized by fusion of a polypeptide to the green fluorescent protein (GFP). Many spectral variants of this reagent are now available, providing powerful tools for studies in living cells. This approach is particularly useful for members of the steroid/nuclear receptor superfamily, since these molecules frequently undergo rapid subcellular redistribution on ligand activation. A major roadblock in the application of this technology concerns problems associated with transient transfections. This technique produces cell populations that are highly heterogeneous with respect to the newly introduced protein and usually contain the protein in a highly overexpressed state. In addition, long-term studies related to cell cycle and cellular differentiation are essentially impossible with this approach. These problems can be overcome by introduction of the GFP fusion into cells under appropriate induction control. We describe application of the tetracycline regulatory system to inducible control of a glucocorticoid receptor (GR)/GFP chimera. Intracellular concentrations of GFP-GR can be very effectively controlled in this system, providing an ideal environment in which to study subcellular trafficking of the receptor and interactions with a variety of intracellular targets.     

Subretinal gene delivery using helper-dependent adenoviral vectors

This study describes the successful delivery of helper-dependent adenoviral vectors to the mouse retina with long term and robust levels of reporter expression in the retina without apparent adverse effects. Since these vectors have a large cloning capacity, they have great potential to extend the success of gene therapy achieved using the adeno-associated viral vector.     

Helper-dependent adenoviral vectors in experimental gene therapy

In the majority of potential applications gene therapy will require an effective transfer of a transgene in vivo resulting in high-level and long-term transgene expression, all in the absence of significant toxicity or inflammatory responses. The most efficient vehicles for delivery of foreign genes to the target tissues are modified adenoviruses. Adenoviral vectors of the first generation, despite the high infection efficacy, have an essential drawback: they induce strong immune response, which leads to short term expression of the transgene, and limits their usefulness in clinical trials. In contrast, helper-dependent adenoviral vectors (HdAd) lacking all viral coding sequences display only minimal immunogenicity and negligible side-effects, allowing for long-term transgene expression. Thus, HdAd vehicles have become the carrier of choice for adenoviral vector-mediated experimental gene therapy, effectively used in animal models for delivery of transgenes into the liver, skeletal muscle, myocardium or brain. Strong and long-lasting expression of therapeutic genes has allowed for successful treatment of dyslipidemias, muscular dystrophy, obesity, hemophilia, and diabetes. Additionally, the large cloning capacity of HdAd, up to 37 kb, facilitates the use of physiologically regulated, endogenous promoters, instead of artificial viral promoter sequences. This enables also generation of the single vectors expressing multiple genes, which can be potentially useful for treatment of polygenic diseases. In this review we characterize the basic features of HdAd vectors and describe some of their experimental and potential clinical applications.     

Characterization of leptin intracellular trafficking

Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR) is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBR) with an intracellular domain of 303 amino acids and a shorter form (OBR) with an intracellular domain of 34 amino acids. Since OBR, is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3-transferrin containing compartments at 37 degrees C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF) and the lysosomal marker protein lamp-1. The transport of leptin was also shown to engage a monensin and bafilomycin sensitive degradation process in lysosomes. Together, our results provide novel data concerning the uptake, intracellular localization and transport of leptin.     

The many highways for intracellular trafficking of metals

Metal ions such as copper and manganese represent a unique problem to living cells in that these ions are not only essential co-factors for metalloproteins, but are also potentially toxic. To aid in the homeostatic balance of essential but toxic metals, cells have evolved with a complex network of metal trafficking pathways. The object of such pathways is two-fold: to prevent accumulation of the metal in the freely reactive form (metal detoxification pathways) and to ensure proper delivery of the ion to target metalloproteins (metal utilization pathways). Much of what we currently know regarding these complex pathways of metal trafficking has emerged from molecular genetic studies in baker's yeast, Saccharomyces cerevisiae. In this review, we shall briefly highlight the current understanding of factors that function in the trafficking and handling of copper, including copper detoxification factors, copper transporters and copper chaperones. In addition, very recent findings on the players involved in manganese trafficking will be presented. The goal is to provide a paradigm for the intracellular handling of metals that may be applied in a more general sense to metals that serve essential functions in biology.Electronic Supplementary Material Supplementary material is available in the online version of this article Abbreviations CTR cell surface transporter - GSH glutathione - MCF mitochondrial carrier family - mito mitochondria - MT metallothionein - SOD superoxide dismutase     

A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein

As the use of adenoviral vectors in gene therapy protocols increases, there is a corresponding need for rapid, accurate, and reproducible titer methods. Multiple methods currently exist for determining titers of recombinant adenoviral vector, including optical absorbence, electron microscopy, fluorescent focus assay, and the “gold standard” plaque assay. This paper introduces a novel flow cytometric method for direct titer determination that relies on the expression of the green fluorescent protein (GFP), a tracking marker incorporated into several adenoviral vectors. This approach was compared to the plaque assay using 10⁻⁴-to 10⁻⁶-fold dilutions of a cesium-chloride-purified, GFP expressing adenovirus (AdEasy+GFP+GAL). The two approaches yielded similar titers: 3.25±1.85×10⁹ PFU/mL versus 3.46±0.76×10⁹ green fluorescent units/(gfu/mL). The flow cytometric method is complete within 24 h in contrast to the 7×10 days required by the plaque assay. These results indicate that the GFU/mL is an alternative functional titer method for fluorescent-tagged adenoviral vectors.     

Encapsulation of adenoviral vectors into chitosan-bile salt microparticles for mucosal vaccination

The objective of this study is the incorporation of adenoviral vectors into a microparticulate system adequate for mucosal delivery. Microencapsulation of the vectors was accomplished by ionotropic coacervation of chitosan, using bile salts as counter-anion. The process was optimized in order to promote high encapsulation efficiency, with a minimal loss of viral infectivity. The maintenance of sterility during all the encapsulation procedure was also taken into account. The principle relies on the simple addition of a solution containing adenoviral vectors to a solution of neutralized chitosan, under stirring. Some surfactants were added to the chitosan solution, to improve the efficiency of this process, such as Tween 80, and Pluronic F68 at 1% (w/v). Encapsulation efficiency higher than 84% was achieved with formulations containing sodium deoxycholate as counter-anion and Pluronic F68 as dispersant agent. The infectivity of the adenoviral vectors incorporated into microparticles was assessed by release assays in PBS and by direct inoculation in 293 and Caco-2 cells. The release in aqueous media was negligible but, when in contact with monolayers of the cells, an effective release of bioactive adenovirus was obtained. Our work shows that encapsulation in microparticles, not only appear to protect the adenovirus from the external medium, namely from low pH, but can also delay their release that is fully dependent on cell contact, an advantage for mucosal vaccination purposes. The formulations developed are able to maintain AdV infectivity and permit a delayed release of the bioactives that is promoted by digestion in situ of the microparticles by the cell monolayers. The onset of delivery is, that way, host-controlled. In view of these results, these formulations showed good properties for mucosal adenovirus delivery.     

Decoding the intracellular trafficking rule for circular RNAs

<正>Circular RNAs(circRNAs) are endogenous single-stranded RNAs with a covalently closed loop generated from 'backsplicing' of precursor mRNA(pre-mRNA)in the nucleus. Mounting evidence emphasizes their significance in cellular homeostasis by serving as miRNA sponges, scaffolds for biomolecules, or templates for translation. circRNA dysregulation has been implicated in human diseases, and clinical trials of circRNAs as potential biomarkers or therapeutic agents are ongoing.     

Endocytosis and intracellular trafficking of ErbBs

This review article describes the pathways and mechanisms of endocytosis and post-endocytic sorting of the EGF receptor (EGFR/ErbB1) and other members of the ErbB family. Growth factor binding to EGFR accelerates its internalization through clathrin-coated pits which is followed by the efficient lysosomal targeting of internalized receptors and results in receptor down-regulation. The role of EGFR interaction with the Grb2 adaptor protein and Cbl ubiquitin ligase, and receptor ubiquitination in the clathrin-dependent internalization and sorting of EGFR in multivesicular endosomes is discussed. Activation and phosphorylation of ErbB2, ErbB3 and ErbB4 also results in their ubiquitination. However, these ErbBs are internalized and targeted to lysosomes less efficiently than EGFR. When overexpressed endocytosis-impaired ErbBs may inhibit the internalization and degradation of EGFR.