부산대학교 도서관

Adeno-associated virus (AAV) vectors are highly efficient tools for use in gene therapy. Current production methods rely on plasmid transfection and are not generally considered amenable to scale-up. To improve recombinant AAV (rAAV) vector production in terms of both final titre and simplicity, we constructed recombinant herpes simplex virus (HSV) vectors, either disabled (ICP27 deleted) or nondisabled, encoding the AAV rep and cap genes. We also integrated an rAAVGFP construct into the nondisabled vector and also into a second pair of HSV vectors (disabled and nondisabled) not expressing rep and cap. Transgene incorporation and expression was confirmed by Southern and Western blot, respectively. Optimal double-infection ratios were established for disabled and nondisabled pairs of rep/cap-expressing and rAAVGFP-containing vectors, resulting in up to 1.55 × 1012 rAAV capsids and 4 × 108 expression units from approximately 1 × 107 BHK producer cells. Functionality of the prepared vector was confirmed by the detection of abundant green fluorescent protein (GFP) expression following injections of rAAV preparations into the rat brain. This paper therefore describes a simple, efficient, and transfection-free rAAV production process based on the use of HSV and not relying on a proviral cell line that, with appropriate scale-up, could yield quantities of rAAV sufficient for routine clinical use. [ABSTRACT FROM AUTHOR]