Canine Adenovirus Vectors: an Alternative for Adenovirus-Mediated Gene Transfer

Abstract

Preclinical studies have shown that gene transfer following readministration of viral vectors is often inefficient due to the presence of neutralizing antibodies. Vectors derived from ubiquitous human adenoviruses may have limited clinical use because preexisting humoral and cellular immunity is found in 90% of the population. Furthermore, risks associated with the use of human adenovirus vectors, such as the need to immunosuppress or tolerize patients to a potentially debilitating virus, are avoidable if efficient nonhuman adenovirus vectors are feasible. Plasmids containing recombinant canine adenovirus (CAV) vectors from which the E1 region had been deleted were generated and transfected into a CAV E1-transcomplementing cell line. Vector stocks, with titers greater than or equal to those obtained with human adenovirus vectors, were free of detectable levels of replication-competent CAV and had a low particle-to-transduction unit ratio. CAV vectors were replication defective in all cell lines tested, transduced human-derived cells at an efficiency similar to that of a comparable human adenovirus type 5 vector, and are amenable to in vivo use. Importantly, 49 of 50 serum samples from healthy individuals did not contain detectable levels of neutralizing CAV antibodies.Human adenovirus types 2 and 5 were chosen as potential gene transfer vectors because of the significant amount of research performed on these serotypes. However, vectors derived from viruses that naturally infect and replicate in humans may not be the optimal candidates for therapeutic applications. Adenoviruses are ubiquitous in all populations and can be lethal in infants and immunocompromised patients (5, 18, 24). More than 90% of the adult population has detectable levels of circulating antibodies directed against antigens from human serotypes (9, 32, 33). Phase I trials using human adenovirus vectors have yielded conflicting results (8,21,41). A difference in humoral immunity that is directed against the vector capsid might explain, in addition to other factors, the variability between and within these studies. Furthermore, when repeat administrations were attempted (7, 42), transgene activity was not detected. Studies aimed at immunotolerization of mice, for the primary or repeat delivery of human adenovirus vectors, are interesting from the immunological standpoint but may have limited practical use in the clinic. Will immunotolerization of patients to adenovirus vectors activate latent, more virulent serotypes? Concomitantly, there are other drawbacks associated with human-derived adenovirus vectors. More than 95% of a healthy cohort had a long-lived CD4 ϩ T-cell response directed against multiple human adenovirus serotypes (14). These data imply that adenovirus serotype switching (27) may have limited advantages. Furthermore, replication-competent adenoviruses (RCAs) (26) can potentially contaminate human adenovirus-derived vector stocks, including gutless adenovirus vectors (16,22), while E1 region-positive vectors are a potential contam...

Osteosarcoma cells as carriers to allow antitumor activity of canine oncolytic adenovirus in the presence of neutralizing antibodies

“…In a similar manner, herpes simplex virus, 17 vesicular stomatitis virus 8 and Newcastle disease virus 29 -infected tumor cells have been reported to activate antitumoral immunity. Previous studies with replication-competent adenovirus-infected carrier-cell systems have reported the use of wild-type HAd5 adenovirus-infected MDA-MG-231 cells 30 and also of granulocyte-macrophage colonystimulating factor-expressing HAd5 loaded into A549 and 293 cells. 10 In this last work, Hamada et al 10 reported that bystander transfection was mediated by carrier-cellderived cell fragments containing viral particles that were engulfed by proliferative target cancer cells.…”

Gutless adenovirus: last-generation adenovirus for gene therapy

“…However, most of them present secondary complications and/or their use in human patients is questionable. These strategies include macrophage depletion, 39,40 use of immunosuppressive agents (cyclosporin A, cyclophosphamide, dexamethasone, FK506, Interleukin-12 and deoxypergualin), [41][42][43][44][45][46] use of antibodies to deplete CTLs, 47,48 blockade of costimulatory interactions between APCs, T and B cells, [49][50][51][52] intrathymic administration of adenovirus, 53 oral tolerization, 54 use of vectors derived from non-crossreacting serotypes, 55,56 use of adenoviruses from other species 57,58 and coating vectors with inert chemicals like polyethylene glycol (PEG). [59][60][61] Diverse in vivo studies in mice suggested that, in the absence of an immune response, first-generation adenoviral vector DNA is maintained as a stable episome in the host cell.…”

“…Different successful strategies to circumvent pre-existing immunity have been applied, such as the use of alternative gutless serotypes 15 and the use of a non-human gutless adenovirus. 58 Thus, administration of gutless CAV-2 vectors allows the stable, high-level expression in neurons throughout the rat central nervous system (CNS). 20 It is noteworthy that even in the presence of an active peripheral immunization with an adenovirus that completely eliminates expression from first-generation vectors within 60 days, gutless vectors are able to maintain a long-term transgene expression in the brain 74,75 due to reduced inflammation both in intensity and in duration in the brains of the preimmunized animals.…”

L’adénovirus canin : meilleur ami de l’homme ?

“…Depuis plus de 6 ans, nous nous intéres-sons à l'adénovirus canin de type 2 (CAV-2). Les vecteurs CAV-2 sont des outils adaptés aux applications cliniques car (1) ils transduisent certaines cellules humaines aussi efficacement que les Ad humains; (2) ils ne se répliquent pas dans les cellules humaines, suggérant un très faible risque de propagation dans l'organisme; (3) une faible ou aucune immunité préexistante n'a été détectée chez les individus testés [2]; et (4) contrairement aux vecteurs dérivés d'Ad humains, le risque de recombinaison et/ou de co-réplication avec les Ad sauvages déjà présents dans l'organisme est quasi nul (pour revue, voir [3]). En outre, le génome de CAV-2 ne s'intègre pas non plus à celui de la cellule hôte ce qui a l'avantage de minimiser le risque de transformation, mais entraîne aussi la perte d'expression du transgène dans des cellules en prolifération, une expression à long terme nécessitant donc, soit des ré-administrations du vecteur, soit le ciblage des cellules post-mitotiques.…”

“…Enfin, si la majorité des Ad utilisent la protéine CAR (coxsackievirus adenovirus receptor) comme récepteur principal et certaines intégrines comme récepteur secondaire (activant des cascades de signalisation qui aboutissent à la production de cytokines pro-inflammatoires [4]), CAV-2 a l'avantage: (1) de ne pas posséder le motif responsable de l'interaction avec les intégrines [5]; et (2) de n'infecter que les cellules exprimant CAR [6], ce qui restreint son tropisme et pourrait permettre de cibler une population cellulaire au sein d'un tissu [7]. In vivo, les vecteurs CAV-2 transduisent efficacement les cellules épithéliales des voies aériennes, in vivo chez la souris ou in vitro chez l'homme [2], encourageant le développement de vecteurs CAV-2 pour traiter la mucoviscidose, maladie récalcitrante à la thérapie génique. L'injection de vecteurs CAV-2 dans diffé-rents tissus a cependant mis en évidence leur tropisme préférentiel pour les neurones [7].…”