I should add that this length includes the AAV ITRs.

To my experience, you could be able to encapsidate your construct but the titer will be lower.

It depends on what serotype of AAV you plan to package into. Some serotypes works better for large insert than others.

I've seen some info on AAV5 being able to package larger inserts and other data to refute that finding. We'll check titers on HT1080 cells before we try with neuronal cultures. Autoimmune response has hampered us before with larger vectors like herpes and lentivirus and so were are placing our bets on the AAV. Any suggestions that could enhance our chances of successfull expression in vivo with an insert this size are appreciated.

Thanks

The largest insert we packaged with AAV5 is ~5.5KB with no affect on the titer.

Do you plan to use AAV5? If so, then you may have trouble testing AAV5 in the HT1080 cells since the infection of AAV5 is very low for this cell. Try CHO or HeLa cells instead for your testing.

Hi,

I am working with AAV quite a lot. I was successful with upto 4.9kb with AAV 2. Alos check the following article:

Effect of Genome Size on AAV Vector Packaging Zhijian Wu1, Hongyan Yang1 and Peter Colosi1 1Ocular Gene Therapy Laboratory, Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health,

Bethesda, Maryland, USA

Hello,

Does anyone know about the smallest size we can put between AAV ITR for an optimal packaging? Is 1000 bp too small? Thanks!

Does anyone have experience with AAV1 as we are currently experiencing problems packaging our vectors of 4.3 kbp?

It's not a problem with the construct size! You might suspect your packaging cells! Use Fresh badge! Or Transfection!

Josephine we routinely package genomes into AAV1 ranging from 4-4.7Kb with no problems and the Samulski lab has shown that AAV1 packages well up to at least 4760bp (Grieger & Samulski 2005). Have you checked that your ITRs are still intact as this can seriously limit vector packaging. The easiest way to do this is to digest your vector plasmid with SmaI prior to transfection as both ITRs should contain 2 SmaI sites. It is very common for one or both of the ITRs to become deleted when AAV plasmids are grown in certain E.coli strains. This inhibits AAV packaging and to minimize this you can grow your plasmids in E.coli strains that limit recombination.

Hi Matt, AAV5 is a suitable choice to package large gene. But, firstly, AAV ITR should be checked for intact, which would affect the yield titer a lot.

Genemedi is expert in AAV production, you could find more information on https://www.genemedi.net/i/aav-packaging