AAV Packaging Frequently Asked Questions
BioInnovatise Viral Vector Team
Updated December 23, 2024
As a viral vector, AAV is in very high demand for numerous reasons. The packaging process for AAV is always customized and executed according to specific research application requirements.
Our viral vector team has successfully produced hundreds of AAV packaging productions for researchers in cell and gene therapy, immunotherapy, and oncology, and along the way, we have addressed many technical questions about AAV packaging. Through these collaborations, we’ve developed extensive expertise in optimizing AAV packaging for diverse experimental needs and therapeutic applications. Here are some of the most common questions we receive from researchers. If you have any questions about your AAV project, please don’t hesitate to contact our viral vector team.
What Is The Best Bacteria For Amplifying Plasmid For AAV Packaging?
Our viral vector team, regardless of the particular vector (adenovirus, AAV, lentivirus, or retrovirus) highly recommends E. Coli for amplifying plasmids. Even for mutagenesis and molecular cloning productions that do not require viral vectors, by default we choose E. Coli.
The most commonly used E. Coli strains for AAV plasmid production are:
DH5α : This strain has low endonuclease activity, prevents recombination, provides high quality plasmid yield, and posses high transformation efficiency.
Stbl3: This strain is particularly important for plasmids containing ITRs because it reduces DNA recombination rates, maintains ITR Stability better than other E. Coli strains, and is suitable for AAV helper and packaging plasmid amplification.
Can You Preform AAV Packaging Without WPRE?
Sure! Many AAV vectors have been successfully assembled and effectively and efficiently delivered genetic payloads to target cells. For those who don’t know, WPRE (Woodchuck Hepatitis Virus) is a regulatory element on an AAV transgene that aims to improve the stability and processing of mRNA int eh nucleus, exchange the export of mRNA from the nucleus to cytoplasm, and increases the efficiency of protein production from the transgene. Altogether these boost transgene expression without taking up more amount of space in the limit cargo capacity of the AAV vector and useful in gene therapy applications with high protein expression is desired.
If you are interested in removing the WPRE element or adding the WPRE element to your AAV transgene, our molecular cloning team can assist.
Can An AAV Package The Entire Dystrophin cDNA?
No, the current AAV packaging capacity (~4.7 kb) is way too limited for the full length cDNA genome which is 14 kb. The two leading workarounds for packaging cDNA in AAV vectors is using fragmented cDNA versions that contain vital cDNA sequences in the packaged vector or using a dual-vector approach which contains larger amounts (yet not the full cDNA sequence) in two AAV packaged vectors.
Can An AAV Package The Entire Dystrophin RNA?
Also no, the entire dystrophin RNA sequence in 14 kb like cDNA. Packaging the entire dystrophin RNA is impossible with a single AAV vector because of the AAV packaging limit of ~4.7 kb.
The two leading workarounds for packaging RNA is the same as cDNA: Using fragmented RNA versions (but translated to DNA) that contain vital RNA sequences in the packaged vector or using a dual-vector approach which contains larger amounts (yet not the full RNA sequence) in two AAV packaged vectors.
If you have additional AAV packaging questions that were not answered, don’t hesitate to reach out to our viral vector team before getting started on your AAV production.
Learn about our quick turnaround AAV packaging services.
Want to learn more about the latest in AAV based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News are always collecting and publishing the latest information on AAV based research.
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