Development of Stable Packaging and Producer Cell Lines for the Production of AAV Vectors

Abstract

Today, recombinant adeno-associated virus (rAAV) vectors represent the vector systems which are mostly used for in vivo gene therapy for the treatment of rare and less-rare diseases. Although most of the past developments have been performed by using a transfection-based method and more than half of the authorized rAAV-based treatments are based on transfection process, the tendency is towards the use of stable inducible packaging and producer cell lines because their use is much more straightforward and leads in parallel to reduction in the overall manufacturing costs. This article presents the development of HeLa cell-based packaging/producer cell lines up to their use for large-scale rAAV vector production, the more recent development of HEK293-based packaging and producer cell lines, as well as of packaging cell lines based on the use of Sf9 cells. The production features are presented in brief (where available), including vector titer, specific productivity, and full-to-empty particle ratio.

Suppression of toxic transgene expression by optimized artificial miRNAs increases AAV vector yields in HEK-293 cells

“…Traditionally, AAVs are either produced in baculovirus-infected Sf9 insect cells 9 or in human embryonic kidney (HEK)-293 cells. Despite increasing successes in the development of stable packaging/producer cell lines (reviewed in Merten 10 ), transiently transfected HEK-293 cells still represent the most commonly used platform for AAV production. Usually, production is based on transient transfection of three plasmids: (1) AAV replication (rep) and capsid (cap) plasmid (rep/cap), encoding genes for genome amplification and viral capsid formation, respectively; (2) helper plasmid (pHelper), encoding adenoviral proteins E2A, E4, and VA for efficient AAV packaging, and (3) an expression cassette-harboring plasmid, minimally existing of a promoter, transgene and polyA signal, flanked by inverted terminal repeats (ITRs).…”