Adeno-associated virus (AAV)

  • Infection of dividing and non-dividing cells
  • several serotypes with different tropisms
  • long-lasting transgene expression, not integrating into the genome
  • 4.7kb DNA uptake capacity
  • ssDNA genome
  • 20-25nm capsid size
  • Security level S1 for recombinant viruses

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AAV vectors and AAV production

For generating adeno-associated viruses you can choose from 3 shuttle vector types. All contain 5' and 3' AAV ITRs and a WPRE. In the basic vector you can express the transgene by using a variety of different promoters. In the moment we are using CamKII, hSynapsinI, CMV early enhancer/chicken β actin (CAG), phosphoglycerate kinase (PGK), EF-1 alpha, CMV and a liver specific promoter (LP1).

In the second set of shuttle vectors we apply LoxP sequences and an invert-oriented transgene to allow specific expression in Cre-transgenic animals. Here the same promoter settings are available as in the first vector type.

In the third set of AAV shuttle vectors we apply the hSynapsin1 promoter together with reporter proteins coupled via a 2A Sequenz to a MCS into which the transgene can be cloned. This configuration allows a bicistronic expression of reporter gene and transgene through one promoter.

We use helper plasmids that provide all the necessary adenoviral genes for AAV replication (E2a, E4, VARNA). To achieve tissue-specific expression, you can choose from a wide variety of AAV serotypes. Currently we can produce serotypes 1, 2, 3, 4, 5, 6, 8, 9, rh10, AAV2retro, AAV9-PHP.B, AAV9-PHP.eB and AAV9-PHP.S. The helper plasmids used all express the AAV2 replicase and the specific capsid proteins for each serotype.

The purification of the AAV particles depends on the selected serotype of the produced AAV. Thus, if desired, heparin columns can be used for AAV2/2 particle purification. Iodixanol gradient centrifugation is used by default for all others. If desired, HPLC purifications for AAV2/8 can be carried out in cooperation with the AG Weger, Institute of Virology of the Charité.

For the serotypes 9, AAV9-PHP.B, AAV9-PHP.eB, AAV9-PHP.S and rh10, Charité external users must have a specific MTA for the helper plasmids with Penn Vector Core. The user receives further explanations during the ordering process.

Costs - AAV Production

Service

Cost

Discription

Regular-scale AAV production

475€ Charité intern / 1000€ Charité extern

Purification of the particles through Heparin-columns, HPLC or iodixanol gradient centrifugation, scale app. 0,5-1ml, 10^10-13 particles / ml

Large-scale AAV production

805€ Charité intern / 1500€ Charité extern

Purification of the particles through Heparin-columns, HPLC or iodixanol gradient centrifugation, scale app. 1-2ml, 10^10-13 particle / ml

AAVs for invitro use

250€ Charité intern / 400€ Charité extern

HEK-cell lysates, No purification, scale app. 0,5-1ml, 10^9-11 particles / ml

     

AAV particel "ready-made"

130€

AAV Particles from our stocks for small scale applications and pilot experiments. Scale 0,1ml 10^10-13 particles / ml (please check the catalog for availability)

Dialysis or buffer exchange after AAV production

80€

AAV Dialysis using "Slide-A-Lyser" or buffer exchange using Amicon filters