FAQ – Adeno-Associated Virus (AAV)

What’s the difference between AAV and Adenovirus?

You can read about the primary differences between the two viral vectors here.

What’s the required biosafety level for using AAVs?

Recombinant AAV constructs in which the transgene does not encode a potentially tumorigenic gene product or a toxin molecule and is produced in the absence of a helper virus can be handled in a Biosafety Level 1(BSL-1) facility. Otherwise, it should be handled as biohazardous material under Biosafety Level 2 (BSL-2) containment.

For more information on biosafety levels, please read the NIH Biosafety Guidelines.

Are recombinant AAVs replication deficient?

For wild type AAV, replication has extremely low efficiency without the presence of helper virus, such as an adenovirus. For recombinant AAV produced nowadays, the replication and capsid genes are provided in trans (in pRep/Cap plasmid).  Only the 2 ITRs of the AAV genome is left and packaged into virion, while the adenovirus genes required are provided either by adenovirus or another plasmid.  Replication of a recombinant AAV is theoretically impossible. This is in similar scheme to lentiviral vectors produced nowadays.

What’s the cloning capacity for recombinant AAVs?

AAV has a packaging capacity of ~4.7Kb. Since the two ITRs of AAV are about 0.2-0.3Kb total, the foreign DNA that can be introduced between these 2 ITRs should be smaller than 4.4Kb, which is much smaller than that of recombinant adenoviruses (7.5Kb). When the length of inserted DNA between the 2 ITRs is close to the maximum allowed (4-4.4Kb), the packaging efficiency decreases significantly. Take gene over-expression from cDNA for instance. Since the CMV-poly(A) element is about 1Kb, the maximal allowable cDNA length is about 3Kb. If you are interested in GFP co-expression (from a separate expression cassette), given the additional CMV-EGFP-poly(A) is about 2 Kb, the maximal cloning capacity for GFP co-expressing system is about 1.0-1.2Kb.

For double-stranded AAV (dsAAV), the capacity is half that of the single-stranded AAV, or ssAAV.

Which AAV serotype and dose should be used for my study?

You can find a thorough review of all serotypes and tissue tropism in our Serotype Selection Guide.

Though AAV has been used extensively for in vivo studies, data regarding which serotype and dose are best for each application/tissue is unclear and sometimes controversial. This is due to different experimental procedures used and end-point readouts. To help you decide which AAV serotype and dose should be used for your application, we have developed an AAV Serotype Testing Kit which contains AAV-GFP for several commonly used serotypes, so that you can test different serotypes side by side in your lab. We advise researchers to do a small-scale, pilot study using these marker viruses.

A route to alter rAAV tropism exploits the natural capsid diversity of other serotypes, such as AAV1, and AAV3-6 etc, by packaging rAAV2 genomes into capsids derived from other AAV isolates. One approach employs hybrid trans-complementing constructs that encode rep from AAV2 whereas cap is derived from the other serotype displaying the cell tropism of choice.

What vector options do you provide for cDNA and shRNA, and what’s their cloning capacity?

For over-expression AAV, you can choose from over 10 different promoters from ubiquitous (CMV, CAG, CBA, EF1a) to tissue specific (Synapsin, CamKIIa, GFAP, RPE, ALB, TBG, MBP, MCK, TNT, aMHC, etc), along with many different fluorescence reporter to co-express (GFP, RFP, mCherry, CFP, YFP, tdTomato, etc). For shRNA silencing, you can choose from Pol III promoters (H1 or U6) and Pol II promoter (CMV) to drive shRNA expression, or a Cre inducible shRNA expression.  Please click here for AAV expression options.

How stable are AAV vectors? How should they be stored?

Stability studies carried out in house and by colleagues show that purified AAV vectors are highly stable at temperatures of 4 C or lower. All viral vectors are shipped frozen on dry ice and should be stored at -80° C upon receipt and for long term storage. Vectors can be stored for short periods of time at -20 or +4°C. Whenever possible, vectors should be aliquoted into single use portions to avoid repeated freeze/thaw cycles. Please aliquot in at least 10ul per tube and use low protein binding tubes to avoid loss of virus.

What’s the difference between physical and genomic particles?

AAV genome particles relate to the viral particles that have been successfully packaged with the genome to be delivered. During the AAV packaging process, many particles that lack the genomic DNA are formed. These particles lack the ability to transduce the cells they come into contact with and are therefore non-functional. As a researcher you want the concentration of functional AAV particles.