iodixanol gradient purification of transfected hek293 cell lysates for concentrated adeno-associated virus production

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

Adeno-associated virus (AAV) was discovered in 1965 and has since been used in a myriad of applications1. AAVs have been applied in neuroscience research to study gene and neuronal function, map neurocircuits, or produce animal models for disease2. Traditionally, this is done by injecting directly at the site of interest, as most natural serotypes do not cross the blood-brain barrier or need a high dose to do so1,2,3.
With the discovery of AAV.PHP.B4 and next-generation capsids such as AAV.PHP.eB5 and AAV.CAP-B106, it is possible to target the central nervous system (CNS) using a simple systemic injection. Spatial mapping reveals the cells targeted by AAV.PHP.eB at cellular level6,7. In combination with specific promoters/enhancers, these capsids offer extensive opportunities for neuroscientists to study genes and brain function by non-invasive AAV delivery4,8.
While a lower dose is needed for AAV.PHP.eB (typically 1 to 5 x 1011 Genome Copies (GC)/mouse) compared to AAV9 (4 x 1012 GC/mouse)7, still more vector needs to be produced compared to direct injection strategies (typically 1 x 109 GC/&#181;L injection). Most natural serotypes can be produced using the classical adherent cell culture system in combination with iodixanol purification9,10,11,12. For AAV.PHP.eB this entails a labor-intensive process to culture and transfect cells to obtain sufficient vectors for one experiment8. Therefore, the production of AAV in suspension cell culture in conical tubes was developed. Conical tubes, with a capacity of up to 300 mL, are compact, saving both incubator space and plastics. Suspension cells are much easier to culture and handle in large amounts than adherent cells on 15 cm plates. The transfection components of the protocol remain the same. Therefore, plasmids previously used with the adherent system can easily be used in this protocol based on production in suspension cells. The protocol was successfully transferred to other researchers in the laboratory and successfully used for various capsids and constructs.

Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Our lab uses viral vectors as a platform to deliver therapeutic genes to the injured nervous system. Recently, numerous non-invasive viral vectors which are able to cross the blood-brain barrier have been developed. Use of these vectors requires high titers and therefore an improved production process.The implementation of our HEK293 Suspension Cell Procedure makes this AAV protocol less laborious compared to other adherent cell based systems, while at the same time gaining higher yields of AV vectors. There are no treatments to repair the damage in the central nervous system. We aim to use non-invasive viral vectors to stimulate central nervous system repair.The advantage of this protocol is that we can now produce viral vectors at high yield, which allows us to perform non-invasive therapeutic gene delivery to the injured central nervous system. Our current work will pave the way to develop non-invasive gene therapy for multiple neurodegenerative diseases such as multiple sclerosis, Parkinson's and Alzheimer's disease.

Iodixanol Gradient Purification of Transfected HEK293 Cell Lysates for Concentrated Adeno-Associated Virus Production

iodixanol-gradient-purification-transfected-hek293-cell-lysates-for

Research

JoVE Journal

Neuroscience

151 Views.  Royal Netherlands Academy of Arts and Sciences (KNAW). Iodixanol Gradient Purification of Transfected HEK293 Cell Lysates for Concentrated Adeno-Associated Virus Production