334 Efficient Gene Transfer in Cultured Aortic Smooth Muscle and Endothelial Cells by Magnetically Responsive Nanoparticles Impregnated with Adenovirus and display high transduction efficiencies in a[.]
and display high transduction efficiencies in a wide range of cell types, both in cell culture and in animals However, data obtained from different studies indicate that gene transfer by Ad vectors is confronted with different hurdles, Onc ofthcsc hurdles is poor transduction efficiency in target cells lacking expression of the primary Ad receptor, CAR (Coxsackie and Adenovirus Receptor) Besides, following in vivo systemic delivery,different tissues are transduced, and particularly the liver, with a high risk of toxicity We first have shown that RGD inelusion in thc HVR5 loop of the hexon monomer (AdHRGD) providesAd with anew integrin-mediated entry pathway in cells in vitro (Vignc ct aI., J Virol, 1999) While performing biodistribution studies in mice, we observed that transgene expression afforded byAdHRGD was dramatically decreased at48h p.i in liver compared with that obtained with unmodified control Ad (Adwt) [n order to analyze whether this decrease was due to RGD motif or to [-[VR5 modification itself, we constructed different Ad containing either Gly-Ala repetitions of different lengths (AdH(GA)8 and AdH(GA)24) or HVR5 sequences from other Ad serotypcs (Ad2, Ad 19 and Ad30) into hexon protein, Following systemic administration in different strains of mice (C57BLl6, BALB/c, C3H), we similarly observeda dramatic decrease (> 95%), in transgeneexpression in liver Iysates, at 48h p.i., whatever the hexon modification was This severe decrease in transgene expression was correlated with an important reduction (84 to 97%) ofAd DNA content in liver as documented by Real-Time Q-PCR However, measurement of liver viral genome content at an earlier time point (30 min) showed a similar accumulation of vector genome in total liver extract for AdH(GA)24 and the control Adwt Thus, vectors containing HVR5 modifications are cleared more rapidly from the liver than Adwt Studies are on their way to examine whether this elimination of viral DNA is linked to a reduction ofhepatocytes transduction and/or to a higher uptake by non-parenchymal cells Besides, we observed that AdH(GA)24 was as efficient as Adwt for transducing tumor cells confirming that Ad infectiousness was not impaired in vivo by hexon HVR5 modification Altogether, our results suggest that enhanced tumor gene transfer could be achieved following insertion of targeting peptides into hexon protein while avoiding a risk of transgene expression into hepatocytes 333 Converging Evidence for a Critical Role of the Diameter of Sinusoidal Fenestrae in Hepatocyte Transduction after Adenoviral Gene Transfer Frank Jacobs,' Eddie Wisse, Yingmei Feng,' Jan Snoeys,' Joke Lievens,' Changchun Ling; Hans Duirnel,' Desire Collen,' Peter Frederik,' Bart De Geest.' 'Center/or Molecular and Vascular Biology University 0/ Leuven, Leuven, Vlaams Brabant Belgium; 2EM Unit Pathology, University Maastricht Maastricht Limburg Netherlands Background: Anatomical barriers constitute a major obstacle for gene transfer The presence of fenestrae in the sinusoidal endothelium is critical for efficient transduction of parenchymal liver cells (PC) Previously, we have demonstrated that strain differences of transgene DNA uptake in PC after adenoviral gene transfer in rabbits correlate with the diameter of sinusoidal fenestrae In addition, interventions that increase the diameter of fenestrae in New Zealand White (NZW) rabbits lead to increased transgenc DNA uptake in PC Objective: The objective of the current study was to provide further evidence for the role of the diameter of fenestrae in hepatocyte transduction Therefore, filtration experiments using filters containing 80 nm, 100 nm and 200 nm pores were performed Second, hepatocyte transduction and the diameter of'fenestrae were compared in NZW rabbits, C57BLl6 mice and Sprague Dawley (SD) rats Third, NZW rabbits were compared with a newly gcncratcd substrain ofNZW rabbits with significantly higher transgcnc SI26 expression Results: Using Vitrobot" technology and cryo-electron microscopy, the diameter of human adenoviral serotype vectors was shown to be 93 nm with protruding fibers of30 nm Filtration experiments demonstrated that the recovery of adenoviral vectors in the filtrate determined by real-time PCR analysis was 0.43 ± 0.091 %,51 ± 7.3 % and 91 ± 1.9 % for a 80 nm, 100 nm and 200 nm filter, respectively Scanning electron microscopy of the filters was in agreement with these data The average diameter of fenestrae determined by transmission electron microscopy in NZW rabbits (103 ± 1.3 nm; n""IO) was lA-fold (p