We also use the 293T cells and definitely get better titers because of the SV-40 large and the SV-40 Ori in our plasmids. I have not noticed a consistent difference between AAV1 and AAV2 titers. Occasionally when something like this pops up it often has to do the quality of the plasmid preps. Is your AAV2 prep older or have you confirmed it recently on a gel? That might be worth considering if the helper plasmid is the only difference.

Hi Jonathan, I thought the name sounded familiar. I hope all is well in Texas. We use a lot of AAV1.SERCA2a in pig models of heart failure. This is also the serotype that was used in a recent clinical trial. Because it is for pigs, we need rather large amount of the AAV1s. We never used AAVDJ8. We consistently get high titers with AAV2, 5, 8 and 9. AAV1 is about 5 to 10-fold lower than these and AAV6 is still worse. There is something very strange going on. I know at least two groups running vector cores that have your experience, i.e. they get better titers with AAV1 than AAV2, and two other vector cores that see the same thing that we see. After talking to one of the groups that has the same experience that you have, the only difference that I could come up with between us and them was the use of 293 vs 293Ts

Hi David, thanks for your answer. I doubt that it is the plasmids. We have done this a million times with different preps of plasmid and even two different plasmid constructs that provide AAV1-Cap, AAV2-Rep and all the Ad helper functions. What plasmid system do you use?

All of our AAV helper plasmids carry AAV2 Rep along with the different Cap sequences. I use pFD6 as an adeno helper and a packaging plasmid that carries the SV-40 Ori to take advantage of the 293T

If you don't mind sharing, what packaging plasmid do you use? Is it published?

Sure, we have published it. its a standard pUC19 backbone with Amp resistance. We routinely change out the promoters and insert target genes in front of the WPRE-BGH poly A sequence. I can send you a plasmid with a poly-linker for cloning target genes if you would like.

Thanks David. Where did you publish it?

We use AAV 293 from Agilent as the packaging cell line for packaging all AAV serotypes. As said earlier in the discussion, AAV1 always gives higher titre when compared with AAV2.

Thanks. What packaging plasmids do you use?

For a viral packaging we use, helper plasmid, a rep and cap containing plasmid and a transgene containing plasmid. EGFP, firefly luc, human factor IX have been some of the most commonly packaged transgenes in our hands.

Thanks. What helper plasmid and what rep/cap system do you use? Especially what is the backbone for your rep/cap plasmids?

The helper plasmid has a pUC ori and contains AdE2A, E4 and VA genes. We use different cap plasmids for different studies but almost all the rep-cap plasmid has AAV2 rep. I am not sure of the backbone of the rep cap plasmid. Guess it must be some cloning vector.

Thanks.

good to know these valuable inputs, I will work on AAV system soon. Thanks, guys.