AAV Titer Determination

Recombinant Adeno-Associated Virus (rAAV) vectors are intensively used for in vivo gene transfer, representing a promising therapeutic approach to target inherited, genetic diseases and many others. Results from animal models and on-going clinical trials show a great potential of AAV vectors with tropism to many different tissues including e.g. the retina, the Central Nervous System (CNS), some internal organs or skeletal muscles. In order to translate these findings to clinical applications it is necessary to approach some concerns.

A major challenge is the potential immunotoxicity of AAV derived gene therapies in form of harmful immune responses against the AAV capsid antigen which might inhibit therapeutic efficacy. The unwanted immune response is suggested to be mainly influenced by the application of empty AAV capsids lacking the transgene of interest. Therefore, it is indispensable to accurately characterize several infectivity-defining measurements like AAV genome titer, infectious titer or the total AAV capsid titer to improve and ensure efficacy and safety of AAV derived gene therapies.

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AAV Titers and Quantification Methods

Different parameters are considered for a comprehensive characterization and specification of AAV vectors, which are a basic requirement according to the FDA guidelines. Beneath the total AAV capsid titer, the AAV genome titer and the infectious titer of the virus preparations, the ratio of full and empty AAV capsids is an important factor concerning the immunogenicity of AAV based gene therapies. The determination of each parameter requires the performance of an individual more or less labor intense technique. 

AAV Genome Titer

physical titer pic

The AAV genome titer [viral genomes per mL, (vg/mL)], also called physical titer is typically measured via quantitative real-time PCR (qPCR), the golden standard method to quantify the concentrations of cDNA or gDNA of a sample2. The relatively fast, easy and cheap technique provides information about the number of capsids containing the transgene of interest with a high through-put. However, several issues including sample processing, dilutions of the standard material, primer design, primer binding, or PCR efficiency can cause high inter-laboratory or inter-assay variations of the results for identical samples1.

Digital-droplet PCR (ddPCR), a more recently emerged DNA detection technique, deals with some of the limitations of qPCR. The principle of this method is the partitioning of the sample into thousands of droplets to separate single DNA molecules and the individual PCR amplification in each subdivision. Since the measurement is performed in one preparation, pipetting errors and contamination problems are minimized. This technique enables a highly sensitive absolute quantification of concentrations without dilution series of standard DNA with known concentrations, as necessary for qPCR5. Unfortunately, beside the high costs for the equipment, variations between labs can still occur due to different sample processing protocols.

Total AAV Capsid Titer

PhysicalTiter pic

The total AAV capsid titer represents the amount of all intact (fully-assembled) viral capsids in a sample, i.e. the full and empty AAV capsids. This parameter is essential for the characterization of AAV vector samples especially regarding the optimization and reduction of the immunogenicity of AAV based gene therapy.

The capsid titer can be determined by the AAV capsid ELISA (enzyme-linked immunosorbent assay) method. PROGEN offers commercially available ELISA kits which have been thoroughly calibrated based on the ATCC standard reference materials (AAV2 & AAV8) or internal gold standards (AAV1, AAV 3, AAV5, AAV6, AAV9, and AAV rh10) representing an easy, cheap and highly trustworthy method for the quantification of AAV total capsid titers. The AAV ELISA kits have been developed based on an exclusive portfolio of capsid-specific AAV antibodies manufactured in-house.

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Alignement AAV5 pic

The AAV total capsid titer can additionally be determined by dot blot. The relatively easy and cheap blotting technique provides semi-quantitative information of the protein of interest lacking the qualitative data that comes along with electrophoresis. For reliable results it is indispensable to use highly trustworthy and specific positive and negative controls as well as highly specific antibodies for the detection of fully assembled AAV capsids. PROGEN provides antibodies, which specifically recognize conformational epitopes in assembled capsids of different AAV serotypes. Hence, they exclusively react with intact AAV particles.

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Ratio of Full versus Empty Particles

ratio full/empty pic

The ratio of full and empty AAV capsids can be determined e.g. by negative stain or cryo electron microscopy (EM). However, the equipment for these techniques is very expensive and sample processing can be very complicated. In addition, the preparation of samples for electron microscopic techniques is known to be prone to cause artifacts, which are likely to distort the final results. Furthermore, these methods are not quantitative methods and therefore are not suitable for absolute quantification of AAV particles (see total AAV capsid titer).

Fortunately, the ratio of full and empty AAV particles can easily be determined by using PCR-based methods, like qPCR or ddPCR in combination with the determination of the total AAV capsid titer via AAV ELISA. The two orthogonal methods can be combined to calculate the ratio from the genomic titer (PCR) and the total capsid titer (ELISA) and define the ratio of full and empty AAV particles in the sample.

Comparison of negative stain electron microscopy (EM) and cryo EM in regard of the full and empty ratio of an AAV3 vector preperation.

Infectious AAV Titer

infectious titer pic

The infectious AAV titer [TU2/pfu3/iu4 per mL] or functional titer provides information about the number of viral particles, which actually transduce the target cells. The determination of the infectious titer can be labor-intense, impractical and often shows high variances as usually observed for cell-based assays.

A frequently used method to assess the infectious titer is the plaque-assay. This assay is based on the cytopathic capacity of the virus and the infection of cells in culture with varying viral dilutions. Infection leads to the lysis of the cells and those in close proximity. This results in small spots (plaques) in the confluent cell mass, which become visible after staining. The number of plaques under consideration of the dilution and viral concentration provide information about the infectious titer [pfu per mL]. However, AAV's lack classical cytopathic effects apart from the helper virus5.

Therefore, another defaulted used method is the cell transduction or transduction unit assay8. One possible proceeding is to attach a fluorophore to the rAAV vector of interest and infect cells in culture. The infectious titer [TU per mL] can be assessed with flow cytometry and the measurement of fluorescent signals in the infected cells8.

In the context of in vivo gene transfer for gene therapy, it is desirable to achieve a good physical-to-infectious particle ratio.

Comparison of AAV Quantification Methods

Each of the mentioned techniques has its pros and cons: qPCR is widely used, but suffers from several issues such as sample preparation, primer design, or PCR efficiency that can lead to high inter-laboratory variation of results for identical samples. Digital droplet PCR methods overcome some of the limitations of qPCR. There is no need of a dilution series of standard DNA with known concentrations to measure an unknown sample, and limited PCR-efficiency is not an issue as it is for qPCR. However, variations between labs can still occur due to different sample processing protocols. Dot blot is a relatively simple and quantitative method, but works only with reliable reference material, while suffering from the limited linearity and dynamic range of western blotting in general.


Given the practical drawbacks of the aforementioned techniques and the importance of the total AAV capsid titer for the development of gene therapies, the integration of a conventional sandwich ELISA into the standard characterization protocol of AAV samples currently appears to be fundamental and indispensable.

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