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