I am trying to design a custom vector (AAV9) from an existing plasmid with an empty MCS. However, the total AAV transgene cassette comes out to just below 5.3kb (my understanding is that you don't really want the size to get about 4.6/4.7kb). If I remove the WPRE sequence, the size goes down to just below 4.7kb. Which is likely a better bet - going with the larger sized AAV or removing the WPRE segment? I leaning towards to the latter.
Thanks
Hi Jamie,
Based on my experience I would guess that even after removing WPRE segment the yield of AAV titre will be quite low and may cause further challenges for your experiment. WPRE element helps to enhance the transgene expression so depending upon the aim of your experiment maybe you can think of alternative options. The link below might be helpful for you in this context.
Chapter Design of AAV Vectors for Delivery of Large or Multiple Tran...
Good luck !
Best wishes
Dear Jamie,
In our experience, and in agreement with Shambhabi's advice, you'll get pretty dismal yields of full-length packaged AAV, if any at all, if you try to work with 5.3kb. Therefore I'd very much recommend you go with your 4.7kb cassette. To enhance expression, you could consider incorporating a short intron and if your total cassette went up to 4.8kb I don't think that would be a major problem.
Late reply but thank Shambhabi Chatterjee and Simon N Waddington for your help!
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