Purification of adenovirus and adeno-associated virus: comparison of novel membrane-based technology to conventional techniques

Abstract

Adenovirus (Ad) and Adeno-associated virus (AAV) are efficient gene delivery systems; manipulation of the wild-type genome allows their use as vectors for the overexpression of desirable transgenes. Generation and purification of such viral vectors can be labour intensive, costly and require specialized equipment, but a new generation of membranemediated ion exchange kits for purification of recombinant virus may facilitate this process. Here, we examine the yields, transgene expression and purity of preparations of Ad and AAV purified using commercially available kits in comparison to other established techniques for purification of recombinant viral vectors. We demonstrate comparable results for Ad and AAV respectively in all parameters investigated, with a substantial reduction in purification time for the kit-based technology. Such approaches are attractive methods for small-scale purification of recombinant Ad and AAV viral vectors. Gene Therapy (2005) 12, S62-S72.

Gene-Modified Mesenchymal Stem Cells Express Functionally Active Nerve Growth Factor on an Engineered Poly Lactic Glycolic Acid (PLGA) Substrate

“…Fourth generation ''gutless'' adenoviral vectors are becoming available. 31,32 The MSCs in our experiments were transduced to express physiologically relevant levels of this growth factor, as demonstrated by ELISA analysis. The concept of ''physiological levels'' of growth factors is not readily modeled in vitro.…”

Immunity to Adeno-Associated Virus-Mediated Gene Transfer in a Random-Bred Canine Model of Duchenne Muscular Dystrophy

“…The two main regimens for purifying AAV vectors include either affinity purification over a heparin column or through a cesium chloride (CsCl) gradient (Duffy et al, 2005;Merten et al, 2005). To determine whether the method of purification affected the magnitude of the cellular immune response observed in our studies, AAV6-CMV-cFIX vectors were purified by either regimen.…”

Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography

“…The use of high-performance liquid chromatography (HPLC) for large-scale adenoviral purification was first described by Huyghe et al [5] and several approaches have been applied, including anion exchange (AIEX), gel filtration (GF), hydrophobic interaction and immobilized metal affinity chromatography [4][5][6]. Unlike traditional processes using CsCl, HPLC offers a straightforward linear scale-up path and procedures for purifying up to around 10 14 input particles have been reported [3,7].…”

“…A variety of culture conditions have been described, ranging from roller bottles, hollow fibre technology and bioreactors involving both perfusion and fedbatch approaches. Purification of viral vectors for research and pre-clinical applications is predominantly performed using a caesium chloride density gradient and overnight ultracentrifugation, although a number of commercially available kits now provide more rapid purification with comparable titre levels [4]. However, these processes are limited to laboratory-scale preparations.…”