Furthermore, both AAV1 and AAV6 have been shown to bind to N-linked sialic acids and removal of sialic acid from the cell surface or competition with lectins inhibits AAV1 and AAV6 transduction.40In this context, it is noteworthy that the majority of secreted proteins are glycosylated and that the N-linked glycosyl models terminate in sialic acid. rabbits showed modest neutralization of AAV transduction whereas porcine sera strongly inhibited transduction by all AAV serotypes and displayed the largest variation between individual animals. Importantly, neutralizing antibody titers as low as 1/4 completely preventedin vivotransduction by AAV9 in rats. Our results suggest that prescreening of animals for neutralizing antibodies will be important for future gene transfer experiments in these animal models. == Introduction == Over the past decade, adeno-associated computer virus (AAV)-based vectors have gained increasing attention in both basic, preclinical and clinical research and AAV vectors are now among the most promising vector systems for gene therapy applications. This is partially due to the fact that AAV is not associated with any known human disease, that AAV vectors can, at least in nondividing cells, generate long-term transgene expressioneven in the absence of genome integrationand that AAVs display relatively low immunogenicity. The relatively low immunogenicity notwithstanding, it has been recognized that this high prevalence of neutralizing antibodies against AAV in the human population presents a considerable obstacle to the broad use of AAV vectors in clinical gene therapy. Whereas the prevalence depends somewhat around the serotype, most studies report that between 20% and 40% of the population has 24, 25-Dihydroxy VD3 neutralizing antibody titers of 1/20 against 24, 25-Dihydroxy VD3 any given serotype.13It is important to point out, however, that it has been 24, 25-Dihydroxy VD3 demonstrated in both animal studies47and clinical trials8,9that neutralizing titers as low as 1/21/4 can prevent successful transduction. Hence, the percentage of patients that need to be excluded from clinical trials is likely substantially higher than the 2040% of individuals reported to harbor neutralizing antibodies against AAV.13 Until now, it was assumed that in animal models neutralizing antibodies against AAVs only play a role in transduction in nonhuman primate models.4,10The presence of neutralizing antibodies against AAVs in nonhuman primates is not unexpected because a large number of AAV variants have been isolated from nonhuman primates.11,12Here, we show that, somewhat surprisingly, neutralizing antibodies against AAVs are present in the sera of several small and large animal models, including mice, rats, rabbits, dogs, sheep, and pigs. The prevalence of neutralizing antibodies depends both around the AAV serotype and the species. Importantly, we show that not only do the pre-existing neutralizing antibodies neutralize transductionin vitrobut alsoin vivoin a rat model as well as in pigs. Taken together, our results suggest that the presence of neutralizing antibodies against AAV, and potentially other serum factors that can neutralize AAV transduction, has to be taken into account when planning and executingin vivoexperiments that use AAV vectors. Based on our data we propose that, especially for large animal models, which use outbred animals and do not come from a germ-free environment, sera of individual animals destined to be included into preclinical studies should be prescreened for the presence of neutralizing antibodies. Furthermore, we propose that animals with neutralizing antibody titers >1/2 should be excluded, at least if the neutralizing antibody titers are decided similarly to the assay described in this manuscript. (For the purpose of this article, we define the neutralizing titers as the dilution at which transduction is usually inhibited by >50% when compared to transduction controls in the absence of serum). == Results == == Pooled mouse serum inhibits transduction by AAV serotypes == During our study of the prevalence of antibodies against AAV1 in a human ITGA11 population we made the surprising discovery that pooled mouse serum inhibits AAV1 transduction (Physique 1a). Strikingly, even mouse serum that was diluted 1/16 inhibited transduction by >50%. Because fetal bovine serum (FBS) also inhibited AAV1 transduction (Supplementary Physique S1), we omitted FBS from the medium for all those our neutralizing antibody assays of sera from other species (Supplementary Materials and Methods). To test whether the surprising inhibition of AAV1 transduction by mouse serum was unique to AAV1 we decided to analyze the effect of pooled mouse serum on transduction by the prototypical AAV, AAV2, 24, 25-Dihydroxy VD3 and by AAV6 and AAV9, both of which are cardiotropic (as is usually AAV1). Interestingly, of these additional serotypes, only transduction by AAV6 was inhibited by pooled mouse serum, whereas transduction by.