Adeno-Associated Virus

Last updated: December 15, 2014



Production of AAV

In the laboratory, Adeno-associated virus is produced by transfecting HEK293T cells with 3-4 plasmids. These plasmids each provide a component or factor needed to produce recombinant virus vectors. After transfection, cell cultures are incubated for 48-72 hrs. At this point, cells are harvested and lysed by sonication and treatment with detergent. Cell lysates are cleared of cell debris and virus is recovered from the supernatant by density gradient centrifugation through an iodixinol gradient. Fractions containing virus are collected from the gradient and put through 2-3 buffer exchanges with artificial cerebral spinal fluid to remove iodixinol and condense the virus. The final preparation is passed through a 200 µM sterile filter. This results in a final virus prep of about 200 µl with titers between 1 X 109-1 X 1011 genomic particles/µl. Genomic particles represent full virus capsids.  The final vectors are defective and do not replicate, even if they are introduced into cells that are infected with herpes virus or adeno virus. 

  • All work is conducted in a biosafety cabinet. 
  • PPE includes gloves, lab coat and a surgical mask. 
  • All waste is autoclaved.

Transport of AAV to Animal Lab

AAV is transported from the lab to LAR in closed cryovials or eppendorf tubes, on ice in an ice bucket with a lid. If virus preps are shipped to collaborators at other institutions, they are placed into eppendorf tubes sealed with parafilm and placed into a 15 ml screw cap tube with KimWipes as absorbent material in case of a spill. The 15 ml tube in placed into a Styrofoam cooler with ice packs. The Styrofoam cooler is placed inside a cardboard box and labeled appropriately.

Use of AAV in Mice

For all studies rAAV1 is injected into the right and left SN.  Anesthetized mice are placed in a David Kopf stereotaxic apparatus (David Kopf Instruments, Palo Alto, CA).  rAAV1 at a concentration of 4 x 109 genomic particles in 4 µl is injected into right and left substantia nigra (SN) of the brain. The  infusion rate is 0.4 uL/minute through a 30 gauge stainless steel needle.  The injection needle is held in place for an additional five minutes to allow uniform diffusion and to avoid return of the liquid through the needle track.  Mice are allowed to recover from surgery and placed in individual cages under the same conditions as prior to surgery.  

  • PPE for surgery involving the use of AAV
    • Lab coat
    • Eye protection
    • Disposable gloves
    • Surgical mask
    • Hair cover
    • PPE for working with AAV-infected mice
      • Lab coat
      • Disposable gloves
      • Surgical mask
      • Clean up and waste disposal
        • Clean up with 10% bleach solution and 70% ethanol
        • Dispose of sharps into dedicated sharps containers
        • Autoclave all waste

Use of AAV in Guinea Pigs

Animals are anesthetized. A small incision is made behind the left ear to expose the bulla. Under sterile conditions a defect is surgically made in the bulla to expose the basal turn of the cochlea. A small hole (chocleostomy) is made in the cochlea and 5µl of virus in delivered via a small catheter at a rate of 0.25µl/min. The cochleostomy is closed with bone wax and the bulla defect is sealed with a small piece of muscle tissue and the incision is closed.

Use of AAV in Rats

Again, under anesthesia and sterile conditions, the skull is exposed and small burr whole is created at stereotactic coordinates. This provides access to the brain. A 33g needle attached to a 10µl Hamilton syringe is slowly placed into the brain and up to 8µl of virus is delivered to the targeted brain region using a mini infusion pump at a rate of 0.5µl/min. After delivery, the needle is withdrawn and the incision is closed. 

Virus is not shed from animals. The virus is quite inefficient and requires a rather high multiplicity of infection (MOI = virus particles/cells) to induce gene expression. A minimum MOI of 10,000 virus particles/cell is needed. Therefore, in the unlikely event that someone pricks a finger with a needle, the amount of virus they would be exposed to is unlikely to induce gene expression. None of the virus preps currently under production involve the use of oncogenic or toxic constructs.

Virus Shedding

Virus is not shed from animals. The virus is quite inefficient and requires a rather high multiplicity of infection (MOI = virus particles/cells) to induce gene expression. A minimum MOI of 10,000 virus particles/cell is needed. Therefore, in the unlikely event that someone pricks a finger with a needle, the amount of virus they would be exposed to is unlikely to induce gene expression. None of the virus preps currently under production involve the use of oncogenic or toxic constructs.

Accidental Exposure

Wash exposed area thoroughly with soap and water for 20 minutes.  Flush mucous membranes with water for 20 minutes.