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FAQ

Frequently Asked Questions

 

Q: Can my gene of interest or expression cassette be packaged into AAV?

A: If your gene of interest is smaller than 4.9 kilo base pairs (kbp), we may package it into our rAAV products. We do not package gene sequences that may be unsafe to humans or the environment, such as those that are carcinogenic and those that encode for dangerous pathogens. If you have any further questions on this topic, you may contact us.

 

Q: What kind of AAV should I use for my tissue- or organ-specific studies?

A: Please see Introduction to AAV for pertinent background information and a useful chart detailing different serotypes of AAV. You may find one to fit your needs.

 

Q: What are the AAV 5.2 and AAV 8.2 products?

A: These AAV products are modifications of wild-type AAV5 and AAV8. In each product, the Phospholipase A2 domain encoded by the VP1 Cap gene has been replaced with the corresponding domain from AAV2. Functionally, the activity of this enzyme allows the virus to escape the cellular vesicle once it has been endocytosed into the target cell, therefore avoiding breakdown by lysozymes. AAV2 demonstrates some of the highest phospholipase activity, so we have integrated the AAV2 wild-type domain into our AAV5.2 and AAV8.2 products with no alteration in tropism to increase infectivity.

 

Q: How do I order pre-made controls?

A: Please request a quote from us. Our controls are produced with a titer of ~2 x 10^13 vg/mL. At this concentration, they will cost $295 per uL, or $2500 per mL. We can schedule ready-made controls for shipment as soon as we receive a form of payment referencing a quote number.

 

Q: How far in advance do you have to schedule an AAV Production project?

A: Our production timeline is 6-8 weeks for custom projects. This timeline will be lengthened if you request a more complex multi-step cloning procedure, or want us to produce your own capsid. We take customer orders daily and will troubleshoot with you if you have any concerns. Please use our newly-designed online ordering system to create a quote through either VTiger or Science Exchange. If you have additional questions, please call us at (510) 887-7121 or email info@virovek.com.

 

Q: What do I send to Virovek before you can initiate my custom project?

A: We require three things: 1) ready-made DNA for your gene of interest, 2) text sequence of your gene of interest, 3) a form of payment. We will sub-clone your DNA into our pFastBac shuttle plasmid so that we may package it in Virovek’s AAV Production System. Please verify that the plasmid DNA you send us matches the text sequence or plasmid map you provide, to avoid delays in your project. See the section titled “Materials Required” on our How to Order page for more specific details.

 

Q: How much plasmid does Virovek need to start a custom project? Does it need to be prepped with an endotoxin free kit?

A: We need 20 uL of plasmid DNA at 0.5 ug/uL at minimum for cloning. If you provide a smaller amount to us, we will spend additional time working on DNA amplification during the cloning step. The DNA does not have to be prepped with an endotoxin free kit. Please see the section titled “Materials Required” on our How to Order page for specific details.

 

Q: I do not have ready-made DNA for my custom project. Can I still use your AAV Production Services?

A: Yes! In this case, we will use a third-party gene synthesis company to create your DNA. There is a cost per nucleotide for synthesis, and we will include this cost in the quote and invoice for your project. Please see this page for more details on plasmid construction and gene cloning.

 

Q: What units does Virovek use to measure production scale and calculate pricing?

A: We use vector genome (vg) as our unit to measure production scale for custom projects. This unit is similar to genome copy (gc) – it refers to the number of complete copies of the customer’s gene of interest that are present in the final suspension. Due to the high-output nature of our AAV generation technology, the lowest production scale that Virovek uses for customer projects is 1 x 10^13 vg.

The titer (in vg/mL), or concentration, of our virus products is typically ~2 x 10^13 vg/mL. It can be adjusted upon customer request.

The volume (in mL) of our virus products can also be adjusted to meet customer needs.

Both titer and volume can be adjusted, but the price of the product will always depend on the vector genome (vg) amount. For example, if you order 1 x 10^13 vg of total virus genome at a concentration of 5 x 10^13 vg/mL, 200 uL of the product will be delivered. The cost will match the production scale, and will be $2500 for academic institutions/non-profits and $4950 for companies.

 

Q: What quality control steps do you perform on AAV vectors? How do you calculate the titer?

A: We do qPCR Titration with our universal ITR primers to quantify vector genome copies, and we are hoping to implement use of a Nanodrop for a more precise quantification of DNA and protein concentation. We also run an SDS Page Gel on AAV proteins called VP1, VP2, AND VP3 to determine purity. These services are offered free of charge and included in our AAV Production Process.

For an additional cost, we can run an endotoxin assay, and/or have the final AAV product sequence-verified.

 

Q: Is AAV safe to work with?

A: Yes, our rAAV is replication-defective, non-communicable, and ready for use in Biosafety Level 1 (BSL-1) settings. We express AAV rep and Cap genes in trans with our unique baculovirus expression system. Baculovirus is specific to insect cells and poses no threat to mammalian cell lines. Our technology allows us to make AAV products without the risk of residual adenovirus or herpes simplex virus contamination (these are both commonly used as helper viruses). Wild-type AAV already lacks many proteins necessary to reproduce on its own, making it a prime candidate for gene therapy research. For more information, please visit Safety and Storage, where you may view our Safety Data Sheet.

 

Q: What is your storage solution for the virus?

A: We use 1 x PBS Buffer containing 0.001% pluronic F-68. The AAV2 capsid will aggregate at high concentrations, so we add sodium citrate to the buffers for these products. If your project features a specialized or novel capsid that is prone to aggregation, we will always consult with you first before deaggregating the product and adding the sodium citrate.

 

Q: How should I store the AAV that I receive?

A: AAV has been proven stable at 4° C and room temperature for use in immediate experiments. For long-term storage, please keep AAV at -80° C for best quality and longevity. Research suggests that storing AAV at -20° C is not sufficient for long-term preservation, and may reduce transduction efficiency of the virus. Be advised that although AAV is a very stable virus, multiple freeze-thaw cycles may reduce the titer of the product. For more information, please visit Safety and Storage and see our AAV Vector Storage and Handling Information.

 

Q: Can I reorder more of the same virus product with the same Lot Number?

A: Yes! Virovek may keep samples and remainders of custom-made AAV for customers up to 4 years after your initial order. Please let us know what Lot Number and volume you would like to re-order, and we will provide you a quote if the product is still available. If you are interested in a titer adjustment, please let us know.

 

Q: How do you pack and ship your AAV products?

A: We pack our AAV tubes in a bubble mailer or bubble wrap in a Styrofoam container and/or an outer cardboard box. The outside of the box will be clearly labeled with Virovek’s logo. For new customers, appropriate protocols on viral transduction will be included in the shipment. We ship parcels through third-party couriers on either ice packs (4°C) or dry ice (-80°C) for a flat rate that is adjusted for distance shipped and temperature control. We encourage you to consider consolidating products from different quotes so we may ship them together in the same package, and waive additional shipping costs.