Adeno-associated viruses

AAV pseudotyping

Adeno-associated virus pseudotyping is the process of combining a capsid and genome from different viral serotypes. We currently have a selection of plasmids for AAV production in stock, purchased from PlasmidFactory. These include pDM and pDG, which are of the wild type AAV2 serotype. We also have a range of pDP series plasmids, which include the rep2 non-structural protein from the AAV2 serotype and various cap proteins. The cap proteins confer different tropisms, depending on which ones are coded.

Production and ordering of AAVs

Production and packaging available for different pseudotypes, which are purchased from PlasmidFactory that hold the license from the German Cancer Research Center. We provide the user with several serotypes to choose from, depending on their needs:

• AAV helper plasmid pDP1 (rep2/cap1)
• AAV helper plasmid pDG (rep2/cap2)
• AAV helper plasmid pDP5 (rep2/cap5)
• AAV helper plasmid pDP6 (rep2/cap6)
• AAV helper plasmid pDP8.ape (rep2/cap8)

The facility has also acquired the necessary plasmids for production of the following pseudotypes:

• AAV helper plasmid
• AAV packaging plasmid PHP.eB
• AAV packaging plasmid PHP.S
• AAV packaging plasmid AAV-DJ Rep Cap

These plasmids enhance the existing pseudotype library available to customers. PHP.eB and PHP.S are based on the AAV9 pseudotype, which facilitates the production of high titer AAV viruses. This enables a less invasive approach for infecting cells in the central nervous system (CNS) using PHP.eB or peripheral nervous system (PNS) using PHP.S. The infection is achieved through systemic inoculation of the virus via intraperitoneal (IP) injection, while still maintaining their specificity to infect only nervous system cells. This is supported by the studies conducted by Chan et al., 2017 and Deverman et al., 2016.

AAV-DJ Rep-Cap plasmid supplies the AAV2 Rep (replication) proteins and the AAV-DJ capsid protein. Recombinant AAV-DJ vectors mediate superior in vitro transduction efficacies in comparison with any other wild type serotypes. Transduction on cell types from different species and tissues, including primary human hepatocytes, melanoma cells, and embryonic stem cells, showed that AAV-DJ vectors were not only superior to all but also substantially better than AAV-2.

Services included

• Transfection and supernatant collection
• Virus precipitation
• Crude purification by centrifugation
• Discontinuous iodixanol gradient ultracentrifugation
• Buffer exchange and concentration with centrifugal filter devices
• Membrane filtration
• Quality control assay include a physical titer (viral genomes (vg)/ml) by RT-PCR or functional titer (IFU/ml) by FACS when suitable. The titration is requested by the customer.

Please note: AAV vector plasmids are known for their instability and frequent deletion of the Inverted Terminal Repeats (ITRs) when propagated in E. coli. Therefore, it’s crucial to thoroughly verify the integrity of each preparation. Even the highest quality plasmid preparations often contain significant amounts (5–15%) of DNA with small deletions in the ITR. These can be randomly selected and amplified during cloning procedures. The integrity of the ITRs is usually monitored by restriction enzyme analysis, with digestion with SmaI or SrfI, which cuts twice within an unstable portion of the ITR. Still the resolution of the agarose gel is not sufficient to detect, for example an 11 bp restriction fragment.

To solve this issue, we recommend using the whole plasmid sequencing service from Eurofins. This service is based on Oxford Nanopore technology that can read GC rich and repetitive sequences (like ITRs) and we have so far positive experience with it.

Furthermore, due to the stability issues, most researchers use a recombination deficient E. coli strain, such as SURE (Agilent Technologies, ref#200152) or NEB stable (NEB, ref#C3040I) for AAV vector plasmid propagation and cloning. Recommended growing temperature is 30°C.

Price list

Higher titer production of up to ≥ 1* 10¹³ vg/ml is possible, please contact us for consultation.

(1) Prices include INDI

(2) From the time the plasmid is received depending on the current queue.

Ordering AAV production

Download the AAV order form. Fill it out and submit to virusfacility@ki.se.