Abstract
AAV vectors have shown great promise for clinical gene therapy (GT), but pre-existing human immunity against the AAV capsid often limits transduction. Thus, testing promising AAV-based GT approaches in an animal model with similar pre-existing immunity could better predict clinical outcome. Sheep have long been used for basic biological and preclinical studies. Moreover, we have re-established a line of sheep with severe hemophilia A (HA). Given the impetus to use AAV-based GT to treat hemophilia, we characterized the pre-existing ovine humoral immunity to AAV. ELISA revealed naturally-occurring antibodies to AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. For AAV2, AAV8, and AAV9 these inhibit transduction in a luciferase-based neutralization assay. Epitope mapping identified peptides that were common to the capsids of all AAV serotypes tested (AAV2, AAV5, AAV8 and AAV9), with each animal harboring antibodies to unique and common capsid epitopes. Mapping using X-ray crystallographic AAV capsid structures demonstrated that these antibodies recognized both surface epitopes and epitopes located within regions of the capsid that are internal or buried in the capsid structure. These results suggest that sheep harbor endogenous AAV, which induces immunity to both intact capsid and to capsid epitopes presented following proteolysis during the course of infection. In conclusion, their clinically relevant physiology and the presence of naturally-occurring antibodies to multiple AAV serotypes collectively make sheep a unique model in which to study GT for HA, and other diseases, and develop strategies to circumvent the clinically important barrier of pre-existing AAV immunity.
Highlights
Adeno-associated viruses (AAVs) have attracted considerable interest in the field of gene therapy because they possess many characteristics that make them excellent vectors for gene transfer
To assess whether sheep harbor naturally-occurring antibodies against the serotypes of AAV commonly employed as gene therapy vectors, Enzymelinked immunosorbent assays (ELISA) were performed on sera from a panel of 6 healthy Merino-Rambouillet sheep using AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 particles as the antigen
The humoral immunity to AAV observed in normal healthy sheep, based on capsid ELISA, mimicked the marked individual-to-individual titer variability of antibodies against AAV capsid that has been observed in the human population [20]
Summary
Adeno-associated viruses (AAVs) have attracted considerable interest in the field of gene therapy because they possess many characteristics that make them excellent vectors for gene transfer. Because genomic integration of AAV vectors is rare [8,9,10,11], the risk of insertional mutagenesis with AAV vectors is greatly reduced compared to retroviruses These collective features have enabled AAV vectors to effectively correct a wide range of diseases in animal models, which, in turn, has prompted numerous clinical trials, in the hopes of safely achieving longterm expression of a variety of therapeutic proteins in human patients. An ongoing clinical gene therapy trial for hemophilia B [12,13] clearly highlights the tremendous potential of AAVbased vectors for the treatment of human disease
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