Adeno Associated Viruses (AAVs)

Overview

Small viruses that can carry relatively small payload, but they do not exhibit pathogenicity nor cytotoxicity in the host organism. They can mediate gene transfer to both mitotic and non-mitotic cells, they lack the risk of oncogene activation, since they can exist stably in an episomal state with a low rate of chromosomal integration. AA virions are small (20-25 nm diameter) and have no enveloped, they carry ssDNA and they are defective virus dependent on the presence of a helper virus for replication.

  • Require BSL2 safety level lab
  • They carry a small payload up to 4.5 Kb
  • No pathogenic and replicant-defective.
  • AAV dsDNA in host cells is maintained in an “episomal” state (not integrated in the host genome).
  • Transgene expression mediate by recombinant AAV vectors can persist for months and up to several years.
  • Can be package with different serotypes that confer different infectious spectrum (tropism):
Serotypes of AAV
Serotype Tropism Comments
AAV1 CNS, skeletal muscle, Heart
AAV2 CNS, photoreceptor cells, Neurons General tropism in all brain areas
AAV4 CNS, Lung
AAV5 CNS, photoreceptor cells, LungAstrocytes, hippocampal neurons Selective retrograde Transport
AAV6 Skeletal muscle, Lung Retrograde transduction
AAV7 Skeletal muscle, Liver
AAV8 CNS, photoreceptor cells, skeletal muscle, Heart, Liver, Pancreas High specificity in Auditory Cortex
AAV9 CNS, skeletal muscle, Heart, Liver, LungNeurons, hippocampus, auditory cortex
A) AAV are non-enveloped, icosahedral and small. They package ~4.7 kB, flanked by ITR singled stranded DNA containing two Open Reading Frames encoding for Replication (rep) and capsid (cap) proteins. B) We currently work with recombinant AAV, meaning that AAV ssDNA no longer contains the cap and rep genes but the transgene which will be introduced in the host cells. Therefore, production of these viruses is achieved by co-transfection of the transgene vector and an AAV helper plasmids that contains the necessary genes for the packaging and virus production within the host cells.

AAV pseudotyping: Define as the mix of a capsid and genome from different viral serotypes. We currently have in stock some plasmids for AAV production purchased from PlasmidFactory including pDM and pDG (Wild type AAV2 serotype), and a series of pDP including rep2 (structural protein from AAV2 serotype) and different cap proteins (capsid protein) conferring different tropisms depending which cap proteins are coded.

Production and ordering of AAVs

Production and packaging available from different pseudotypes. Purchased from PlasmidFactory which holds 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 acquired the necessary plasmids for production of the following pseudotypes:

  • AAV helper plasmid
  • AAV packaging plasmid PHP.eB
  • AAV packaging plasmid PHP.S

These plasmids expand the current pseudotype library at the customers disposal. These pseudotypes are based on the AAV9 pseudotype that allows the production of high titer AAV viruses that allows a less invasive method of the CNS (PHP.eB) or PNS (PHP.S) cell infections by systemic inoculation of the virus via IP while still retaining their specificity to infect only Nervous system cells (Chan et al., 2017; Deverman et al., 2016).

Services include:

  • Transfection and supernatant collection
  • Virus precipitation.
  • Crude Purification by centrifugation, discontinuous Iodixanol gradient and anion exchange using adsorption membrane.
  • Buffer exchange and concentration by centrifugation filter devices
  • Filtration
  • Quality control assay include a physical titter (vg/mL) by RT-qPCR or functional titter (IFU/mL) by FACS when appropriated. The titration is requested by the customer.

Note: AAV vector plasmids are notorious for the instability and frequent deletion of the ITRs when propagated in E. coli. For this reason, it is important to carefully confirm the integrity of every preparation. Even the best plasmid preparations contain significant quantities (5–15%) of DNA containing small deletions in the ITR, which during cloning procedures can be randomly selected and amplified. The integrity of the ITRs is typically monitored by restriction enzyme analysis sufficient to identify small deletions (20 bp). Most commonly this is achieved with digestion with Sma I or Srf I, which cut twice within an unstable portion of the ITR. Because of these stability issues, most investigators utilize 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.

Material provided and price list:

~500 µl in chosen size aliquots, titer ≥1010 vg/mL (viral genomes/mL). Prices include titration by qPCR or FACS:

Physical titration: 10.000 SEK/virus for KI users; 12.000 SEK/virus for external users.

Functional titration (if available): 9.500 SEK/virus for KI users, 11.000 SEK/virus for external users.

Ordering AAV production

  1. Download the order form
  2. Fill it out and submit to: aisha.ahmed@ki.se

Links

AddGene AAV Guide
Lentiviral Vectors

Lentiviral Vectors

The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression, are attributes that bring lentiviral vectors to the forefront of gene delivery vehicles for research.

Gamma-Retroviruses

Gamma-Retroviruses

The Gammaretrovirus is a sister genus to the lentivirus clade. Like the lentivirus, it is a member of the Orthoretrovirinae subfamily of the retrovirus family, they share common properties but the main difference between these two viruses is that Gammaretroviruses only infect dividing cells.