RESEARCH ARTICLE Genetic manipulations of adenovirus type 5 fiber resulting in liver tropism attenuation E Vigne 1,2 , J-F Dedieu 1,2 , A Brie 1,2 , A Gillardeaux 1,2 , D Briot 1,2 , K Benihoud 1 , M Latta-Mahieu 1,2 , P Saulnier 3 , M Perricaudet 1 and P Yeh 1,2 1 UMR1582 CNRS/IGR/Aventis, Institut Gustave Roussy, Villejuif, France; 2 Gencell S.A., Vitry-sur-Seine, France; and 3 Centre de re ´fe ´rence IGR/Aventis/Gencell, Institut Gustave Roussy, 94805 Villejuif Cedex, France The development of genetically modified adenoviral vectors capable of specifically transducing a given cell population requires the addition and functional presentation of particular tropism determinants within the virus capsid, together with the abrogation of the molecular determinants that dictate their natural tropism in vivo. The human adenovirus serotype 5 (Ad5) first attaches to the cell surface following high-affinity binding of the C-terminal knob of the fiber capsid protein to the coxsackie and adenovirus receptor (CAR). Here we have assessed whether genetic shortening of the fiber shaft (virus BS1), or replacing the Ad5 fiber shaft and knob with their Ad3 counterparts (virus DB6), could cripple this interaction in vitro and in vivo. A 10-fold decrease in the binding of the modified capsids to soluble CAR was evidenced, which correlated with a similar reduction of their ability to transduce CAR-positive cells in vitro. The ability of BS1 to interact with cellular integrins was also impaired, suggesting that the penton base and the short-shafted fiber when embedded in the capsid preclude each other from efficiently interacting with their cognate cell surface receptors (CAR and integrins respec- tively). BS1 and DB6 intravenous injections in mice further supported a profound impairment of the ability of the capsid- modified viruses to transduce the liver as demonstrated by a 10-fold reduction of intracellular viral DNA and transgene expression. Interestingly enough, the host humoral response was also specifically weakened in BS1- and DB6-inoculated animals. Taken together, these observations indicate that (i) fiber shortening and (ii) pseudo-typing of Ad5-based vectors with the shaft and knob from non-CAR-binding serotypes constitute two promising strategies to successfully attenuate their native tropism in vitro and most importantly in vivo. Gene Therapy (2003) 10, 153–162. doi:10.1038/sj.gt.3301845 Keywords: adenovirus vector; targeting; tropism; fiber Introduction Redirecting adenovirus (Ad) vectors to specific cell types has become a major challenge in recent years. 1 Restrict- ing gene delivery to a desired cell population indeed constitutes a promising approach to increase both the safety and efficacy of adenovirus-mediated gene transfer in vivo. First, systemic or loco-regional injections are likely to be safer, due to an abolished or reduced virus uptake by non-target tissues. For example, cellular binding and internalization of Ad5-based vectors typi- cally trigger a cascade of signaling events, including upregulation of NFkB, IL-8 or various chemokines, 2–7 which likely contribute to the acute inflammatory and injury responses that characterize this class of vector in vivo. 2–4 Second, targeted vectors are likely to be less immunogenic provided their uptake by antigen-present- ing cells (APCs) is significantly reduced as proposed. 8 Finally, it is widely recognized that systemic administra- tion of Ad5-based vectors results in a rapid and almost exclusive transduction of the liver in mammals, prevent- ing efficient delivery of the therapeutic transgene to other compartments. 9–12 The ability to genetically control the virus tropism in vivo will certainly expand the use of adenoviral vectors beyond loco-regional administrations in humans. The coxsackie and adenovirus receptor (CAR) has been previously identified as high-affinity attachment receptors for Ad5-based vectors. 13,14 Virus binding to the cellular surface is followed by its internalization in clathrin-coated vesicles, an event that is mediated by the specific recognition of an RGD motif in the penton base by av integrins. 15,16 The deciphering of the intimate structure of the viral capsid, together with a thorough understanding of the virus–cell molecular interactions 17– 22 has opened the way to the development of capsid- modified adenoviruses displaying a novel tropism. In particular, several groups have recently demonstrated the permissivity of particular domains within the fiber and hexon proteins for functional presentation of small peptide ligands at the capsid surface, thereby extending the repertoire of cell types susceptible to Ad5 uptake and infection. 23–27 On the other hand, strategies aiming at abrogating the virus native tropism have proved more challenging. Whereas deleting the fiber gene from the adenoviral genome certainly appears as the most straightforward approach to circumvent virus attach- ment to the cell surface, this approach has met with limited success as the modified capsids were reportedly immature and unable to escape the endosome Received 6 March 2002; accepted 16 June 2002 Correspondence: E Vigne, Gencell S.A., CRVA, 13 quai Jules Guesdes, 94403 Vitry-sur-Seine, France Gene Therapy (2003) 10, 153–162 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt