Abstract
Recombinant adeno-associated viruses (rAAV) are promising candidates for gene therapy approaches. The last two decades were particularly fruitful in terms of processes applied in the production and purification of this type of gene transfer vectors. This rapid technological evolution led to better yields and higher levels of vector purity. Recently, some reports showed that rAAV produced by transient tri-transfection method in adherent human embryonic kidney 293 cells can be harvested directly from supernatant, leading to easier and faster purification compared to classical virus extraction from cell pellets. Here, we compare these approaches with new vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies. This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability.
Highlights
Recombinant adeno-associated viruses are used as tool for gene transfer applications
Nonionic detergent action highly simplifies Recombinant adeno-associated viruses (rAAV) purification process Original rAAV purification process from tri-transfected HEK293T cells has recently evolved by using supernatant rather than cell pellet to recover viral particles.[20]
It is important to note that the Total cell culture (TC) approach proposes a more ergonomic activity since it uses fewer stages before the 0.45 μm filtration process that ends all the clarification steps
Summary
Recombinant adeno-associated viruses (rAAV) are used as tool for gene transfer applications. Clinical trials, using rAAV as gene therapy vectors, have yielded promising results[1] and first marketing approval, using a rAAV1 vector, was delivered by the European Union in October 2012 for the treatment of familial lipoprotein lipase deficiency.[2,3] These vectors belong to the Parvoviridae family, nonenveloped viruses embedding a single-stranded DNA genome packaged into a 25 to 30 nanometer icosahedral shaped capsid These last two decades offered large panel of upstream and downstream methods for rAAV, aimed at constantly improving the yields, the purity, and the scale and the time of the processes.[4]. Iodixanol-based isopycnic ultracentrifugation is generally preferred to the classical CsCl2 approach due to its higher particles recovery yields.[10,11] Chromatographic methods, using either ion-exchange[12–14] or affinity-based approaches are used.[15–17] unlike ultracentrifugation, chromatographic techniques are more serotype specific and need more technical adjustments to separate the full from empty particles
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