AAV vectors have received a great deal of attention for clinical gene therapy (GT), since they transduce many mitotic and quiescent cells and mediate long-term transgene expression. Unfortunately, many of the serotypes of AAV commonly employed in GT procedures ubiquitously infect humans, generating pre-existing immunity against the AAV capsid proteins that precludes efficient transduction or induces CTL responses to the transduced target tissue. At present, highly successful animal studies have not translated into clinical success in humans, due, at least in part, to the paucity of animals which harbor endogenous antibodies which recognize and bind AAV-2 and other AAV serotypes for which humans are the natural host. Sheep have long been used as a model to study a broad range of disease states, and a high degree of clinical predictability has consistently been observed. We therefore examined whether sheep possess antibodies to AAV and could thus serve as a much-needed preclinical model system for evaluating AAV-based GT. ELISAs were performed on sera from a panel of 6 healthy Merino-Rambouillet sheep using AAV-1,-2,-5,-6,-8, -9 particles as the antigen. Our results demonstrate that sheep naturally harbor antibodies to all 6 AAV serotypes tested, yet the titers against the different serotypes varied greatly from sheep-to-sheep. While one sheep exhibited very high level (>2300ng/ml) IgG against all 6 AAV serotypes tested, others exhibited moderate/low (>350ng/ml) IgG against all 6 AAV serotypes, and still others exhibited moderate/low level IgG against only 3–4 of the tested serotypes. Despite these differences, all sheep harbored detectable antibodies to AAV 2, 5, & 8. A luciferase-based neutralizing antibody (NAB) assay was then performed on sera from 3 of the sheep exhibiting the highest titer IgG against AAV 2, 8, & 9 to assess the clinical significance of these antibodies in the context of AAV-based GT. All 3 animals harbored relatively high titer (1:100-1:316) NAB to AAV 2, but only 1 animal harbored significant NAB titers against AAV 8 & 9 (1:31, and 1:100, respectively). B cell epitope mapping of these 3 animals with a library of peptides derived from the capsids of AAV 2, 5, 8, and 9 revealed that each individual sheep harbored antibodies recognizing from 17 to 50 of the various capsid-derived peptides, some of which were common to all capsids, and some of which were unique to specific AAV serotypes. Importantly, many of the identified capsid epitopes have also been shown to be recognized by antibodies present in human patients with existing AAV immunity. To our knowledge, this is the first report of an animal disease model harboring naturally occurring functional antibodies to serotypes of human AAV commonly employed as GT vectors. The close parallels between human and sheep physiology, coupled with our recent re-establishment of sheep with severe hemophilia A with a null mutation in the FVIII gene and the presence of these antibodies, suggest that sheep may represent an ideal large animal model system in which to study GT in the context of pre-existing immunity to AAV, and to develop novel strategies for circumventing this immunologic barrier.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.