422. Optimization of AAV Vector Production by Transient Transfection

Abstract

Recombinant adeno-associated virus (rAAV) vectors hold promise for human gene therapy. Although most preclinical studies and current phase I clinical trials use vectors derived from rAAV serotype 2, other serotypes have been identified which transduce certain cell types more efficiently. The availability of sufficient quantity and quality of rAAV vectors is a key element in obtaining reliable and reproducible data and is essential for the transition to human clinical trials. Furthermore, to allow a fair comparison of the transduction efficiencies of rAAV vectors of different serotypes in vitro and in vivo, their quality and purity should be similar. This stresses the need for a universal purification method for different AAV serotypes. The aim of this study was to develop such a method, making use of single-domain antibody fragments from species of Camelidae. These antibodies have excellent binding characteristics, unusual high physical and thermal stability due to their single domain nature, and are efficiently produced in S.cerevisiae. A single domain antibody fragment with affinity for rAAV2 was selected from a naive Llama phage library. The selected antibody fragment was coupled to chromatography resin and displayed high affinity for rAAV vectors pseudotyped with capsids of serotype 1, 2, 3, and 5 when tested in small scale chromatography experiments. These results indicated that the single chain antibody fragment recognized a conserved epitope in the capsid proteins of these serotypes. The process was further optimized and scaled up to allow purification of clinical grade rAAV1 vector. This resulted in a simple, reproducible, and efficient purification process providing highly purified rAAV1 with recoveries of approximately 50%. Characterization of the purified rAAV1 in vitro and in vivo, demonstrated that this vector induced good levels of transgene expression. From this study we conclude that single domain antibody fragments are extremely powerful for efficient purification of rAAV vectors and will significantly improve the characterization and comparison of rAAV vectors of different serotypes as vectors for gene therapy applications.