Factors that may affect AAV tropism?

02 February 2018 5 9K Report

Hi all,

We've noticed that our AAV (serotype2) has gradually changed tropism over the years. AAV2 purified 10 years ago had high tropism for neurons with little glial transduction, whereas now days, we're getting a lot of glial background. Titers have not changed significantly. Two possibilities that we can think of are:

1. We've changed purification protocol. Originally we used the Virapur AAV purification kit. Now we use Sepharose AVB with pH 3.0 elution (and immediate neutralization to pH 8.0).

2. Our FBS serum lot has changed.

How much of an impact do you think these would have on tropism? Has anyone had similar experience?

EDIT: I want to clarify, the AAV we are purifying more recently seems to have broader tropism, whereas the AAV purified years ago showed more specific tropism. So, it isn't a matter of degradation during storage.

Jai Ghosh

The kit change has virtually no effect on tropism. Your second point is not clear i.e what do you mean by saying that FBS serum lot has changed. Elaborate a little as to what is that significant change there. Possibly storage for 10 years have brought about the change which is glial background. Finally, it appears that it does not interfere with ypour experiments, so why break-heads so much.

Kelly Kawabata

Regarding FBS, I was wondering if variation in serum may somehow affect packaging/post translation modification of VP1/2/3, which would in turn affect tropism. Its a well known phenomenon that serum can vary widely in trace components, which in turn can affect cell metabolism, viability, etc. I was curious if anyone observed similar effects.

Jai Ghosh

You need not apprehend that variation in serum would affect tropism regardless of the variation in trace components. It might affect cell metabolism but not tropism.

Kelly Kawabata

Thank you for your answer. Do you have any idea why the AAV we produce now may more widely transduce cells (glial/neuron ratio increases), compared to before, despite the changes mentioned above in our protocol? This change in tropism is actually a major problem due to the increased background fluorescence, making it difficult to image our neurons of choice.

Jai Ghosh

Therefore, it was a technical fault on your part...

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