Hello! I currently observe discrepancies between qPCR titers and in vivo efficiency of our AAV2/1 and AAV2/5 injected into the brain. My qPCR quality controls are good (no contamination, amplification(X2)ok, external control ok, standard cruve at R=0.999, etc...). So I am wondering if contaminating proteins may affect "infectivity" of these AAVs. A gel of capsid proteins revealed a good staining of VP1; VP2 and VP3 but some contaminating proteins...
Hi Marie,
I am not totally sure whether I understand your question. Do you mean you measured high AAV titers but do not see efficient transduction in vivo?
There might be multiple possible reasons for that... Have you checked protein expression mediated by your AAVs in vitro, i.e. by transducing cells in culture? This might give you an idea of the overall bioactivity of your AAV batches. Also, in case you would like to directly compare AAV1 and AAV5, you should ideally quantify them on the same qPCR run to compensate for normal fluctuations between different runs.
Anyway, regarding your question whether contaminating proteins in AAV preps can impair transduction efficiency: I'd rather say no, if they are not massive. People have used low purity AAVs for many years (because of a lack of good purification methods) and they still worked... There is even a publication (Tenenbaum L, Hamdane M, Pouzet M, et al. Cellular contaminants of adeno-associated virus vector stocks can enhance transduction. Gene Ther 1999;6:1045–1053) claiming that contamination with host cell proteins can enhance transduction.
Nevertheless, highest vector purity is of course very desirable, especially when it comes to CNS administration, as contaminating proteins might trigger inflammation.
Thank you very much Benjamin, you answered to my question ! :)
For additional info, I have titrated AAV5 and AAV1 with the same PCR. My titers are apparently high, around 8.06E+14 and 6.28E+13 vg/mL respectively. Despite these high titers, I have had to inject them non diluted (2microL) into the brain, to see a signal. I found it really surprising, as I already injected 10 to 100-fold less (of an AAV1, titered and made in another lab) to see the same quantity of the protein of interest (evaluated by ELISA dosage). For the moment, I can not compare AAV1 and AAV5 efficiency in vitro, because we have only HEK cells and AAV5 do not transduce HEK cells...Do you know a cells that I can use to evaluate
...I am probably over-estimating titers in qPCR titration... But I have checked : My quality control are OK (a RSS8 as an internal control that is titrated as 2 fold less than the theorical value, the DNase for removing non-encapsiated DNA before qPCR is ok, water show no contamination, standard curve ok, replicability ok...), BUT the melting curve indicates a first peak before the peak corresponding with the amplicon ! => On the agarose gel: only one amplicon is detectable ! ( my primers are universal ITR primers, and my amplicon is located into the ITR... I was wondering if the tridimensional structure of the ITR amplicon may produced signal as it contains a lot of complementary structures and may probably refolding on itself, generating signal with sybergreen ? ... But at 60°C (annealing t° used in then PCR) , is it really happening ? I mean... Apparently this kind of folding may occur for temperature lower than 60°C...
As you suggest, I also believe that you probably heavily overestimated your titers. I would consider 10^13 a "normal" range, but with batches exceeding 10^14 at lab production scale I would be kind of sceptical.
I can't really speculate on your ITR structure-related theory, however, I would suggest to try a different primer set, e.g. binding within the promoter region. We have made very good experience with that. Also, I don't know whether you use one specific ITR-containing plasmid to prepare a standard curve? If so, you might also want to try using the exact plasmid that you used for the production of your AAVs as the reference plasmid. Anyway, qPCR-based quantification is a topic for itself and, as you are probably aware, several groups have published work on the efficiency and accuracy depending on the method used, primer binding sites, different standard material, etc
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