What information do you need from me to produce my vector?
Please download the AAV Research Grade Production Order form. Please include the following information:
- AAV serotype to be produced.
- Promoter name and source (e.g., human, mouse, viral, etc.).
- Transgene name and source (e.g., human, mouse, etc.).
- Biological activity of transgene.
- PolyA sequence (if applicable).
How do I prepare my AAV cis plasmid for shipping?
- Purify your plasmid using an endotoxin-free kit.
- Send at least 1200 μg of plasmid packaged with ice packs, with the tube well shielded from breakage.
- Verify the integrity of the inverted terminal repeats (ITRs).
- Verify the integrity of the expression cassette using restriction enzyme mapping and/or sequencing.
- Optional, but recommended: Do a transient transfection of your plasmid into an appropriate cell line to confirm transgene expression and/or function.
Can you send me a cis cloning plasmid that I can use to create the vector plasmid?
- Yes, we have cis cloning plasmids into which you can clone your cDNA or your expression cassette.
What are the limitations on the transgene?
- The total amount of sequence that can be packaged into an AAV is approximately 4.8 kb.
- The ITRs take up approx. 300 bp, leaving about 4.5 kb of foreign sequence to be inserted.
- The exact size of transgene depends on the size of your promoter, polyA, and any other sequences you include (such as an intron).
Can I use self-complementary AAV (scAAV)? If so, what are the limitations?
scAAV (also known as double-stranded AAV (dsAAV)) is produced from cis plasmids containing a deletion in one of the two terminal resolution sites (trs). It can be provided to customers who have a research license to the technology. If you are interested in scAAV’s please provide proof of your research license with your order. Because scAAV is double-stranded, its genome size is approximately half the size of that packaged in single-stranded AAV, corresponding to approximately 2 kb of foreign sequence.
Can you provide vectors with standard reporter genes?
Vector Core can provide vectors expressing lacZ or EGFP from the ubiquitous CMV promoter.
Which grade of AAV vector production is available?
At the moment only the production of research grade AAV batches is operational. For the production of GMP grade AAVs (GMP manufacturing and release of AAV batches for phaseI/II clinical studies) the facility is under construction. The objective is to get the GMP facility authorized for the production of IMPDs by the Italian Drug Agency (AIFA) by the end of 2022.