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Frequently asked questions

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FAQ about the AAV-PHP capsids

AAV-PHP use and biodistribution

Does AAV-PHP.B spread better than other AAVs when injected directly into the brain parenchyma? 

AAV-PHP.B and AAV9 appear to spread to a similar degree based on injections into the mouse striatum. In our unpublished data, there was no obvious difference in tropism or spread when injected directly into the brain. 

Does the route of IV administration (retro-orbital sinus or tail vein) affect the biodistribution of the virus? 

Not to our knowledge. We use retro-orbital sinus injections on the advice of our veternarian staff and because it's easier and more reliable in our hands. Based on data from other groups, tail vein injection appears to provide similar vector distribution. 

We would like to express our gene of interest in astrocytes specifically, would you recommend AAV-PHP.A or AAV-PHP.B? 

We strongly recommend using AAV-PHP.B. Together with the GfABC1D promoter developed by Michael Brenner (U. of Alabama), AAV-PHP.B will provide efficient and selective gene expression in astrocytes (see efficiency data in Deverman et al 2016). AAV-PHP.A, while more selective for astrocytes, requires higher virus doses and is extremely difficult to work with because the virus produces poorly (AAV-PHP.A yields are typically ~30X lower than AAV9) and because it also aggregates quickly in PBS. We have not figured out how to store this virus without significant (visible) aggregation. 

Does AAV-PHP.B work in species other than mice? 

Experiments to test this are underway. AAV-PHP.B appears to provide increased CNS transduction, relative to AAV9, in young rats, and in collaboration with Nina Huber and Sergiu Pasca we showed that AAV-PHP.B transduces human neurons and glia in cortical spheroids (see Supplementary Fig. 5). We have heard from several groups that AAV-PHP.B does not transduce the CNS of songbirds following IV injection. 

Is AAV-PHP.B specific for the brain, or does it transduce other organs? 

AAV-PHP.B transduces the CNS as well as many other organs. Please see Deverman et al 2016 for biodistribution data and images of several organs following PARS-based tissue clearing. 

How much AAV-PHP vector do we need to inject? 

This depends on your experimental needs and the specific cell population(s) you wish to target. You will likely have to test this experimentally. We are happy to provide guidance to help you get started. 

How do you perform IV injections in adult mice? 

We access the vasculature in mice via the retro-orbital sinus. Please see this helpful paper for a protocol and a discussion of the benefits of this route versus tail vein injections. 

Virus production

What are the packaging capacities of the AAV-PHP vectors? 

We have not tested this systematically, but we do not expect they differ from other natural AAV capsids. We have achieved high titer preparations of genomes around 4.7kb or slightly larger. As expected, genomes larger than 5kb have not packaged well. 

Will our current AAV9 preparation method work with the PHP.B serotype?

The PHP.B serotype is slightly more prone to loss (possibly due to aggregation) during the prep as compared to AAV9. This should not be a problem if the purification is done following the protocol which we provide. It may work with other protocols, but we cannot guarantee this. Once purified, AAV-PHP.B can be concentrated to very high titers (>1e14 vg/ml) and the yields are similar to AAV9. Note, we lyse the cells in a high salt buffer. While this step may not be essential, it may reduce the risk of aggregation during purification. 

Should we use a standard pHelper plasmid (expressing E4, E2A and VA-RNA) together with AAV-PHP.B rep-cap?

Yes, use a standard AdV helper. 
Does the recombinant AAV plasmid have AAV2-type ITRs. Did you try with a self-complementary AAV genomes?
Yes, use AAV2 ITRs. We do not find it necessary to use scAAV genomes to achieve rapid, efficient tranduction of the CNS via the vasculature. If your vectors are scAAVs, there is no reason to think they should package less well in the engineered PHP capsids than in natural AAV capsids.
How does the use of the iCAP-PHP plasmids differ from the use of a standard AAV rep-cap plasmid? Do I need to add doxycycline during virus production?
The iCAP-PHP plasmids have a tTA-TRE based inducible amplifiction loop built in to increase virus production. In our hands, these iCAP plasmids increase virus production by nearly 2-fold as compared to standard rep-cap plasmids when used at the transfection ratios provided in our protocol. This system does become complicated if your genome has a tet responsive element, such as with the use of our TRE containing VAST constructs. In this case the tTA on the iCAP plasmid will drive high level expression from the TRE containing AAV genome, which will reduce virus production.  For most of our AAV-PHP variants, we can provide a the capsid in a standard rep-cap plasmid format. 
It seems 1 - 2 x 10^12 vector genome/mouse is needed for efficient transfection of neurons (reporter as GFP). What is the volume of this amount of virus for IV injection?

This depends on the titer of the virus, but it is best to keep the injection volume between 30-150 ul for injections in mice.

What is the concentration of your virus after purification? 

This varies from prep to prep, and from genome to genome. An optimal yield post purification is 2-4E12 vg/150 mm dish of producer cells. 

Is there anything particular about your using PEI for cell transfection? Will lippofectamine 2000 do the job too?  That cause less cell toxicity, right?

We’ve never tested lipofectamine. Its use would add signifiant cost to this procedure given the scale of transfection. Also, there may be something particularly advantagous about PEI transfections because the transfection efficiency of 293 cells is lower than with Ca2+ phosphate, but the virus yields are nearly 10x higher, at least in our hands.

In the protocol that you sent us on packaging and purification of AAV, I did not find information about the quantity and ratio of AAV capsid, AAV vector, and AD helper for the transfection of packaging cells. Could you please provide some information on this?

We use a vector: capsid: helper ratio of 1:4:2 based on ug of DNA. We use 40 ug of DNA total for each 150 mm dish. 

Obtaining reagents

How do we request plasmids? 

Please see our addgene page (coming soon). You can also email Ben Deverman (bd at caltech dot edu) and Viviana Gradinaru (viviana at caltech dot edu) with your request. 

Can we have a trial sample of one of your capsids? 

Given the large volumes and titers of AAV-PHP vectors required for IV administration, we are unable to keep sample stocks of the PHP viruses on hand at all times. If we have stock available, we are happy to send some to you. Please email Ben Deverman (bd at caltech dot edu) and Viviana Gradinaru (viviana at caltech dot edu) with your request. 

Are there commercial sources of the AAV-PHP capsids? 

There are companies selling AAV-PHP.B. We have not worked with any of these companies to validate their vectors. 

Will you make a custom virus prep for us? 

We are happy to discuss your project with you and come up with the best solution for your vector needs. We don't yet offer custom virus preparations as a service, but we do produce vectors for other groups on a collaborative basis. 



FAQ about CREATE

We would like to use CREATE in our own lab, can you provide the plasmids and a protocol? 

Yes, we are happy to share the plasmids, a protocol, and advice to help you use CREATE. Please email us. 


 

FAQ about VAST

What is VAST?

See the discription and application of VAST here: http://rdcu.be/tIcC and on our AAV page.  FAQs and more information coming soon.