Marcelo Simon Sola
rAAV vectors platform
Publish at 20.11.2019
The aim of the rAAV platform is to include larger scale production and to open this facility to all the Institute Imagine teams as well as other research institutes.
The production system was put in place based on a published technology that has several advantages in terms of flexibility of manufacturing multiple serotypes, yields up to ~1E14 vg per run, purity and potency. The same protocol is used for purification of all AAV vectors regardless of serotype.
The facility is able to produce rAAV vectors for expression of therapeutic transgenes, reporter genes (GFP, luciferase), empty capsid particles (AAV particles with no genome inside) as well as rAAV vectors associated with extracellular vesicles (exo-rAAV).
The tissue culture capability is up to 100 roller bottles (RB) per week, allowing a timely production of small and large amounts of vectors to support both research studies as well as small and large pre-clinical animal studies.
Vector purification was improved and streamlined, for example the clarification of the crude cell lysate allows the direct visualization of the vector bands on the cesium chloride (CsCl) gradient used for the vector purification. AAV preparations are extremely pure, empty capsid-free, with minimal content of protein and nucleic acid contaminants.
Cells are transfected with helper virus-free, triple plasmid transfection method for AAV vector production (Matsushita et al 1998; Wright et al 2009; Xiao et al 1998).
- Roller bottle incubator with 100 bottles capacity/run