Pharmacological interventions for improving adenovirus usage in gene therapy.

Gene therapy may be an innovative and promising new treatment strategy for cancer but is limited due to a low efficiency and specificity of gene delivery to the target cells. Adenovirus is the preferred gene therapy vector for systemic delivery because of its unparalleled in vivo transduction efficiency. Intravenous administration of low doses of adenovirus results in adenovirus sequestration in the liver due to binding to the scavenger receptor present on Kupffer cells. When the amount of adenovirus surpasses the binding capacity of Kupffer cells, hepatocytes absorb adenovirus particles in a blood factor-dependent manner. Increasing the Ad dose even more will saturate both the Kupffer cells and hepatocytes. Then sinusoid endothelial cells bind adenovirus particles in an RGD motif-dependent manner. Strategies to eradicate the binding to liver cells include drugs to interfere or eliminate binding to specific cell types, adenovirus capsid protein mutations and chemical modifications of adenovirus to shield the capsid proteins from cellular receptors. The combined use of these approaches should ultimately lead to successful systemic application of adenovirus in humans.

Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes.

During heart development, cells from the proepicardial organ spread over the naked heart tube to form the epicardium. From here, epicardium-derived cells (EPDCs) migrate into the myocardium. EPDCs proved to be indispensable for the formation of the ventricular compact zone and myocardial maturation, by largely unknown mechanisms. In this study we investigated in vitro how EPDCs affect cardiomyocyte proliferation, cellular alignment and contraction, as well as the expression and cellular distribution of proteins involved in myocardial maturation. Embryonic quail EPDCs induced proliferation of neonatal mouse cardiomyocytes. This required cell-cell interactions, as proliferation was not observed in transwell cocultures. Western blot analysis showed elevated levels of electrical and mechanical junctions (connexin43, N-cadherin), sarcomeric proteins (Troponin-I, alpha-actinin), extracellular matrix (collagen I and periostin) in cocultures of EPDCs and cardiomyocytes. Immunohistochemistry indicated more membrane-bound expression of Cx43, N-cadherin, the mechanotransduction molecule focal adhesion kinase, and higher expression of the sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a). Newly developed software for analysis of directionality in immunofluorescent stainings showed a quantitatively determined enhanced cellular alignment of cardiomyocytes. This was functionally related to increased contraction. The in vitro effects of EPDCs on cardiomyocytes were confirmed in three reciprocal in vivo models for EPDC-depletion (chicken and mice) in which downregulation of myocardial N-cadherin, Cx43, and FAK were observed. In conclusion, direct interaction of EPDCs with cardiomyocytes induced proliferation, correct mechanical and electrical coupling of cardiomyocytes, ECM-deposition and concurrent establishment of cellular array. These findings implicate that EPDCs are ideal candidates as adjuvant cells for cardiomyocyte integration during cardiac (stem) cell therapy.

Selective degradation of peroxisomes in yeasts.

In the last two decades, much progress has been made in understanding the process of induction and biogenesis of peroxisomes, essential organelles in all eukaryotes. Only relatively recently, the first molecular studies on the selective degradation of this important organelle-a process known as pexophagy, which occurs when the organelles have become redundant-have been performed, especially using methylotrophic yeasts. The finding that pexophagy and other transport pathways to the vacuole (vacuolar protein sorting, autophagy, cytoplasm-to-vacuole-targeting and endocytosis) utilize common but also unique genes has placed pexophagy in the heart of the machinery that recycles cellular material. The quest is now on to understand how peroxisome degradation has become such a highly selective process and what the signals are that trigger it. In addition, because the prime determinant of pexophagy is located on the peroxisome itself, it has become essential to study the role of peroxisomal membrane proteins in the degradation process in detail. This review highlights the main achievements of the last years.

Selective targeting of adenovirus to alphavbeta3 integrins, VEGFR2 and Tie2 endothelial receptors by angio-adenobodies.

Tumor angiogenesis is a prominent mechanism, driving the development and progression of solid tumors and the formation of cancer cell metastasis. Newly formed tumor vessels represent an elective target for the activity and the delivery of cancer therapeutics. We targeted adenovirus (Ad5) to endothelial receptors which are up-regulated during the formation of new blood vessels, to enhance the efficiency of anticancer gene therapy applications. Bifunctional angio-adenobodies were constructed by the fusion of a single chain antibody directed against the adenoviral fiber knob, to different peptides recognizing the alpha(v)beta(3) integrins, VEGFR2 and Tie2 receptors on endothelial cells. The angio-adenobodies were coupled to the adenoviral vector, containing luciferase and GFP as reporter genes. In vitro data showed selective targeting of the Ad5 to the endothelial receptors both in mouse (H5V) and human cell lines (HUVEC). H5V cells, refractory to Ad5 infection, showed high level of luciferase expression when cells were infected with targeted virus. Viral transgene expression increased in HUVEC cells when cells were infected with Ad5 conjugated with angio-adenobody thereby demonstrating the affinity of the peptides for human endothelial cells also. In vivo data obtained from mice bearing a C26 colon carcinoma subcutaneously show viral transgene expression only in tumors infected with angio-adenobodies retargeted adenovirus. The results of the present study demonstrate that endothelial targeted angio-adenobodies represent a versatile tool to direct adenovirus from its native receptors to the integrins alpha(v)beta(3), VEGFR2 and Tie2 receptors that are fundamental in many angiogenesis related diseases such as cancer.

Scavenger receptor A: a new route for adenovirus 5.

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.

Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages.

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.

ALG2, the Hansenula polymorpha isocitrate lyase gene.

To set the basis for molecular and cellular studies of the glyoxylate cycle in methylotrophic yeasts, we isolated and characterized ALG2, the Hansenula polymorpha isocitrate lyase gene. Complementation work and sequence analysis revealed an ORF of 1458 nucleotides, encoding a 486 amino acid protein with a predicted molecular mass of 54.9 kDa. This protein is shorter than the Saccharomyces cerevisiae and Candida tropicalis ICLs, lacks a PST1 signal and possesses a PTS2-like signal. The transcriptional regulation of ALG2 mRNA levels by carbon source is mainly achieved by glucose repression-derepression, whereas ethanol induction plays only a minor role. We present evidence indicating that, in H. polymorpha, neither isocitrate lyase activity nor the ALG2 gene product are necessary for C(1)-peroxisome degradation triggered by ethanol. Therefore, the involvement of glyoxylate in degradation, as described by Kulachkovsky et al. (1997) for Pichia methanolica, does not necessarily apply to all methylotrophic yeasts. The relevant nucleotide sequence has been deposited at GenBank (Accession No. AF373067.1).

Cold-inducible selective degradation of peroxisomes in Hansenula polymorpha.

Exposure of Hansenula polymorpha cells, grown in batch cultures on methanol at 37 degrees C, to a cold treatment (18 degrees C) is paralleled by a rapid degradation of peroxisomes present in these cells. Remarkably, the events accompanying organelle degradation at 18 degrees C are similar to those of selective glucose-induced peroxisome degradation in wild-type cells, described before. This observation was strengthened by the finding that cold-induced peroxisome degradation was not observed in mutants impaired in selective peroxisome degradation (Atg(-) mutants). Biochemical data indicated that the onset of peroxisome degradation was not triggered by the inactivation of peroxisome function due to the fall in temperature. We show that our findings have implications in case of fluorescence microscopy studies that are generally not conducted at physiological temperatures and thus may lead to strong morphological alterations unless proper precautions are taken.

Removal of Pex3p is an important initial stage in selective peroxisome degradation in Hansenula polymorpha.

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.