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

Summary

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Abstract

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 10¹¹ genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 10¹² genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 10¹² GC in total, and TransIt yielded 2.4 x 10¹³ GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Introduction

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 so^1,2,3.

With the discovery of AAV.PHP.B⁴ and next-generation capsids....

Protocol

All experimental procedures were approved by the institutional animal care and use committee of the Royal Netherlands Academy of Sciences (KNAW) and were in accordance with the Dutch Law on Animal Experimentation under project number AVD8010020199126. In Figure 1, a schematic overview of the complete protocol is provided. From seeding cells to AAV purification, the protocol takes 6 days to complete.

1. Reagent preparation

1. Plasmid purif.......

Discussion

Systemic administration of AAV is a powerful tool for gene transfer to the CNS; however, the production of AAV is an expensive and laborious process. By using suspension cells, labor and plastics are reduced compared to the adherent culture of HEK293T on 15 cm² plates. Furthermore, the conical tubes implemented here are easy to handle and maximize the use of laboratory space. The protocol was set up by two researchers and subsequently applied by others in the lab. A series of productions by three independ.......

Materials

Name	Company	Catalog Number	Comments
39 mL, Quick-Seal Round-Top Polypropylene Tube, 25 x 89 mm - 50Pk	Beckman Coulter	342414
Adapter 600 mL conical tubes, for rotor S-4x1000,	eppendorf	5920701002
Adapter Plate fits 16 bioreactors of 600 ml	Infors HT/ TPP	587633
Aerosol-tight caps, for 750 mL round buckets	eppendorf	5820747005
Centrifuge 5920 R G, 230 V, 50-60 Hz, incl. rotor S-4x1000, round buckets and adapter 15 mL/50 mL conical tubes	eppendorf	5948000315
Distilled Water	Gibco	15230147
DNase I recombinant, RNase-free	Roche	4716728001
DNase I recombinant, RNase-free	Roche	4716728001
DPBS, calcium, magnesium	Gibco	14040091
DPBS, no calcium, no magnesium	Gibco	14190144
Fisherbrand Disposable PES Filter Units 0,2	Fisher	FB12566504
Fisherbrand Disposable PES Filter Units 0,45	Fisher	FB12566505
Holder for 50 ml culture tubes also fits falcon tube	Infors HT/ TPP	31362
Holder for 600 ml cell culture tube	Infors HT/ TPP	66129
Incubator Minitron 50 mm	Infors HT	500043
LV-MAX Production Medium	Gibco	A3583401
N-Tray Universal	Infors HT/ TPP	31321
OptiPrep - Iodixanol	Serumwerk bernburg	1893
PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)	Poly-sciences	24765-100
Phenol red solution	Sigma-Aldrich	72420100
Poly(ethylene glycol) 8000	Sigma-Aldrich	89510
TransIT-VirusGEN	Mirus	Mir 6706
Trypan Blue Solution, 0.4%	Gibco	5250061
TubeSpin Bioreactors-50ml	TTP	87050
TubeSpin Bioreactors-600ml	TTP	87600
Viral Production Cells	Gibco	A35347
Vivaspin 20 MWCO 100 000	Cytvia	28932363