Remember the game show "The $25,000 Pyramid" where one player tries to get the other to guess a category by listing the things that belong in that category? Alright let's play. I list the examples and you try to guess the category: Any ideas? That's right... Headline WaysVAA!
ELISA...
qPCR...
Droplet digital PCR...
ADN-Dotblot...
Transduktion assay...
PAGE FDS.
Electron microscope...
The point is that there are many ways to quantify the composition of avector viralsolution, and in general these methods measure different properties of the solution. These characteristics tell us about three important factors.
What can AAV titles tell you?
- physical title: the concentration of viral particles containing viral genomes. Physical titers are measured by quantifying the concentration of viral genomes (usingqPCRor other DNA quantification methods - see below), since each viral particle normally contains a viral genome. Except for a theoretical maximum value, physical titers are not necessarily indicative of infectious titer. The infectious titer of a viral vector strain can vary depending on the transduction target and can also be altered by freezing and thawing the viral solution (Reliquary al., 2010).
- infectious titer: the concentration of viral particles that cells can transduce. Infectious titers are normally quantified by cell transduction assays. Wild-type AAV2 has been reported to have a nearly perfect 1:1 ratio of physical to infectious particles (Zeltner et al., 2010). However, for recombinant AAV2, the same study reported a ratio of physical to infectious particles of 50:1 (Zeltner et al., 2010). The specific infectivity of virus preparations is defined by the ratio of physical virus particles to infectious virus particles.
- Ratio of full to empty viral capsids: the proportion of genome-containing viral particles relative to the total number of viral capsids (which may include empty capsids that do not have a viral genome). Empty viral capsids can be formed during viral production depending on the experimental conditions used to generate AAV. One study showed that about 50% of viral capsids were genome-containing ("full") (Zeltner et al., 2010), while another reported that only 20% of recombinant AAV2 particles were filled compared to 50% of wild-type AAV2 pools (Grimmet al., 1999). The percentage of genome-containing viral capsids is typically quantified by electron microscopy of a viral vector solution. Because this technique is intensive and requires an electron microscope, it is not routinely performed on all new viral vector preparations. Addgene used this method to extensively validate our viral vector preparation protocol and we are pleased to report that more than 95% of the viral capsids in representative viral vector preparations are genomic (Figure 1).
*Thanks to David Bell and Svetla Stoilova-McPhie, Center for Nanoscale Systems, Harvard University.
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Figure 1: Electron micrograph of an Addgene AAV viral vector preparation after negative staining. Empty vector particles can be identified after negative staining and appear darker than complete vector particles. This image shows that the vast majority of vectors are complete particles (white arrowhead) versus empty particles (black arrowhead). Scale bar = 100nm. |
What title do people usually give?
Addgene and other viral vector manufacturing sites report the physical titer of a viral solution (Figure 2). Since physical titers are used for dosing purposes in preclinical studies, it is important to understand what these values mean and how they can be compared.
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| Figure 2: Label on aliquots of Addgene AAV. Our titer values (circled in red) indicate the physical titer as measured by qPCR. |
The physical titer is usually calculated by two common PCR-based methods:
- Quantitative PCR (qPCR)
- Digital droplet PCR (ddPCR)
In the past, a quantitative DNA hybridization (DNA dot blot) method was used to titer AAV, but this method is not widely used today.Fagonet et al., 2012).
PCR-based methods are robust, simple, fast, and convenient. However, these methods are not necessarily precise or accurate for AAV quantification, as PCR can be affected by many experimental factors. To list some factors that complicate AAV quantification:
- The efficiency of qPCR can be influenced by the second-order structure of the AAV genome caused by repeats and self-complementarity of the AAV genome and ITRs.
- The efficiency of qPCR can be influenced by temperature parameters. For example, we found that changing the PCR annealing temperature from 60°C a 61°C improved the reliability of the trial.
- Different primers can have different annealing efficiencies (Wang et al., 2013). Although you can optimize your primers for each individual sample, this reduces convenience and comparability as each sample must be quantified with a different primer pair.
- Ct values will vary depending on the amount of starting material in the PCR, as the sample must be within the linear range of the standard curve. In addition, high and low concentration samples can also behave differently in PCR, even if both samples are within the linear range of the assay. This may be due to competition for binding sites between primers and AAV genome repeats.
- Primer annealing can be affected by the presence of protein contaminants, which is a factor in AAV titer, since the starting template is the purified viral vector solution (containing viral capsid proteins) rather than DNA purified. This can be mitigated by using purified viral DNA as a template instead of the intact AAV particle.
- Finally, assay precision for the final titer value can vary up to two-fold simply due to assay noise. In other words, repeating the exact same PCR twice can produce titer measurements that vary by as much as two times.
How can we achieve a more reliable AAV qualification?
For ourviral servicewe are currently evaluating AAV vector preparations by qPCR. To ensure accurate and reliable results, we have optimized our assay in several ways:
- Absolute quantification by qPCR requires the creation of a standard curve of known concentration. We regularly create and validate new plasmid standards.
- We validated our absolute quantification using the AAV Universal Reference Standard Material (AAV SFM), an AAV sample that has been quantified by 16 laboratories worldwide and can be used by researchers to validate their qPCR assay (Reliquary al., 2010). In addition to the AAV-RSM, we include a second known-titer reference AAV sample that has a higher titer than the AAV-RSM. By using these samples in all of our qPCR assays, we ensure that the generated standard curve is reliable and can therefore be used to accurately derive titer values for new AAV samples.
- We also typically quantify our AAV sample via two qPCR assays and compare the values for precision and reliability. In doing so, we assess whether the two titer values are within assay error and therefore considered reliable, or whether the sample needs to be requantified.
Some of the issues in qPCR titration are also addressed.ddPCR technology(Hindson et al., 2011,Hindson et al., 2013), which has been shown to be more accurate and repeatable than qPCR (Taylor et al., 2017). However, since purified AAV preparations contain low and similar levels of background protein and chemicals, ddPCR may not be an improvement over qPCR (Taylor et al., 2017).
What do the Addgene AAV titers mean for your experiments?
Since AAV (physical) titers are approximate and highly dependent on the experimental conditions used, titer values of samples from different sources should not be compared. If you are using AAV from different sources in the same experiment, consider thisRename both AAVsin your lab. Absolute title values may not be as important as relative title values. Finally, since the physical titer is not indicative of an infectious titer, you should consider titrating each batch of AAVliveto determine the optimal dose. We at Addgene do not reserve bundles, but if you have a backorder for the same virus, feel free to email us and we will do our best to get you a specific bundle (if we still have it available).
references
1.Fagone, Paolo, et al. "Systemic errors in quantitative polymerase chain reaction titration of adeno-associated self-complementary viral vectors and improved alternative methods".Human gene therapy, Part B: Methods23.1 (2011): 1-7. PubMedPMID:22428975. PubMed CenterPMCID:PMC3640491.
2.Grimm, D., et al. "AAV-2 Particle Titration via a Novel Capsid ELISA: Genome Packaging May Limit Recombinant AAV-2 Production".gene therapy6.7 (1999). PubMedPMID:10455443.
3. Hindson, Benjamin J., et al. "High-throughput droplet digital PCR system for absolute DNA copy number quantification".Analytic chemistry83.22 (2011): 8604-8610.4. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, Vessella RL, Tewari M. Absolute Quantification by droplet digital PCR versus analog real-time PCR. Nat-Methoden. 2013 de octubre; 10 (10): 1003-5. PubMedIDPM:22035192. PubMed CenterID de PMC: PMC3216358.
4.Blockade, Martin et al. "Characterization of a recombinant adeno-associated virus type 2 virus standard reference material".human gene therapy21.10 (2010): 1273-1285. PubMedPMID:20486768. PubMed CenterID de PMC: PMC2957240.
5.Taylor, Sean C., Genevieve Laperriere, and Hugo Germain. "Digital Droplet PCR versus qPCR for gene expression analysis with few abundant targets: from nonsense variables to publication-quality data".scientific reports7 (2017). PubMedPMID:28546538. PubMed CenterID de PMC: PMC5445070.
6.Wang, Feng et al. "A reliable and feasible qPCR strategy for AAV vector titration".Medical science oversees basic research19 (2013): 187. PubMed.PMID:23828206. PubMed CenterID de PMC: PMC3706409.
7.Zeltner, Nadja, et al. "Near-perfect infectivity of wild-type AAV as a benchmark for the infectivity of recombinant AAV vectors".gene therapy17.7 (2010): 872-879.PubMedPMID:20336156. PubMed CenterID de PMC: PMC2900506.
Additional resources on the Addgene blog
- Important considerations when using AAV
- Addgene AAV Quality Control Methods
- look at oursFeatured topic page of viral vector
Resources at Addgene.org
- Find all available AAVs
- learn more aboutvirus production
- MeetViral vector protocols
Subjects:viral vectors,Viral Vectors 101,VAA