Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the liver, Kupffer cells are responsible for adenovirus degradation and contribute to the inflammatory response. As scavenger receptors present on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated the possible implication of these receptors in the clearance of the adenovirus vector. Polyinosinic acid [poly(I)], a scavenger receptor A ligand, was analysed for its capability to inhibit adenovirus uptake specifically in macrophages. In in vitro studies, the addition of poly(I) before virus infection resulted in a specific inhibition of adenovirus-induced gene expression in a J774 macrophage cell line and in primary Kupffer cells. In in vivo experiments, pre-administration of poly(I) caused a 10-fold transient increase in the number of adenovirus particles circulating in the blood. As a consequence, transgene expression levels measured in different tissues were enhanced (by 5- to 15-fold) compared with those in animals that did not receive poly(I). Finally, necrosis of Kupffer cells, which normally occurs as a consequence of systemic adenovirus administration, was prevented by the use of poly(I). No toxicity, as measured by liver-enzyme levels, was observed after poly(I) treatment. From our data, we conclude that poly(I) can prevent adenovirus sequestration by liver macrophages. These results imply that, by inhibiting adenovirus uptake by Kupffer cells, it is possible to reduce the dose of the viral vector to diminish the liver-toxicity effect and to improve the level of transgene expression in target tissues. In systemic gene-therapy applications, this will have great impact on the development of targeted adenoviral vectors.
Selective degradation of peroxisomes (macropexophagy) in Hansenula polymorpha involves the sequestration of individual organelles to be degraded by membranes prior to the fusion of this compartment with the vacuole and subsequent degradation of the whole organelle by vacuolar hydrolases. Here we show that Pex3p, a peroxisomal membrane protein essential for peroxisome biogenesis, escapes this autophagic process. Upon induction of macropexophagy, Pex3p is removed from the organelle tagged for degradation prior to its sequestration. Our data indicate that Pex3p degradation is essential to allow the initiation of the organellar degradation process. Also, in a specific peroxisome degradation-deficient (pdd) mutant in which sequestration still occurs but the vacuolar fusion event is disturbed, the turnover of Pex3p is still observed. Taken together, our data suggest that degradation of Pex3p is part of the initial degradation machinery of individual peroxisomes.Peroxisomes are important organelles that play a role in various metabolic processes in eukaryotes. In fungi, they are predominantly involved in the oxidative metabolism of the carbon and/or nitrogen source used for growth. Characteristically, during cultivation on compounds that require the function of peroxisomal enzymes, the organelles rapidly develop and contain the key enzymes involved in the metabolism of the specific growth substrate. The opposite also occurs. When cells grown at peroxisome-inducing conditions are placed in fresh media in which these functions are no longer required for growth, the organelles are rapidly and selectively degraded by an autophagic process, also termed pexophagy (1-2).In Hansenula polymorpha, selective degradation of peroxisomes is induced when methanol-grown cells are shifted to fresh glucose-or ethanol-containing media. This process involves three morphologically distinct steps, namely (i) sequestration of the organelle to be degraded from the cytosol by various membranous layers, (ii) the fusion of the outer membranous layer of the sequestered compartment with the vacuolar membrane followed by (iii) the degradation of the organellar components by vacuolar hydrolases (3). This process has been designated macropexophagy.In the related species Pichia pastoris, a similar process takes place after a shift of cells from methanol to ethanol. However, when glucose is used to induce pexophagy, an alternative degradation pathway is initiated termed micropexophagy. The hallmark of this process is that clusters of peroxisomes are engulfed by the vacuole (4).Recently, we observed that the mechanisms of peroxisome biogenesis and selective degradation in H. polymorpha use factors in common. We demonstrated that Pex14p, a crucial component in matrix protein import, is also essential for selective peroxisome degradation in H. polymorpha (5,6). Although the molecular mechanisms of the dual function of Pex14p in the two oppositely directed processes are still unresolved, we showed that the information that controls degradation resides in t...
Adenoviruses are common pathogens associated with respiratory diseases, gastrointestinal illnesses and/or conjunctivitis. Currently, this virus is used as a vector in gene therapy trials. The promise of viral gene therapy applications is substantially reduced because the virus is cleared by liver macrophages upon systemic administration. The mechanism underlying adenoviral tropism to and degradation in macrophages is poorly understood. We identified a new adenoviral receptor, the scavenger receptor A (SR-A), responsible for uptake of the virus in macrophages. CHO cells expressing SR-A showed increased viral transgene expression when compared with wild type cells. Preincubation of J774 macrophage cells with SR-A ligands decreased significantly adenoviral uptake. Electron-microscopy analysis of infected J774 cells showed activation of a viral degradation pathway. Infection of mice with adenovirus resulted in a substantial decrease of the virus in liver macrophages when SR-A was blocked. Our data provide a basis for understanding of the adenoviral uptake and degradation mechanism in macrophages in vitro and in vivo. Inhibition of adenoviral SR-A uptake can be utilized in gene therapy applications to increase its efficiency and efficacy.
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