The Problem with Comparability

A major issue for analytical characterization

The community of gene therapy is experiencing an exponential increase in the number of clinical trials of which most are clinical trials being conducted in the USA. This increase in the gene therapy field also gives rise to a great number of companies specializing in the production of gene therapies for the treatment of genetic disorders. A major challenge for the community as well as the regulatory authorities is the understanding of the thresholds for the analytical characterization needed to evaluate the safety of gene therapies.

AAV-based gene therapies reach from small dose applications to high doses depending on the target organ or tissue. However, finding the right dose for a specific gene therapy represents a major question which needs to be answered during the clinical trials. While the dose of the therapy is supposed to be high enough to allow the best possible treatment it is important to evaluate the lowest dose possible to avoid severe side effects that might accompany the delivery of the transgene. Since AAV-based gene therapies are limited to a single application choosing the right dose is a critical step. Beside the contribution of pre-existing conditions, principal issues in transferability of data from animal experiments as well as comparability of meanigful titers might represent additional difficulties for the evaluation of the safety of a specific product. The dose of AAV gene therapies is usually defined by the copy number of vg/kg. However, it is known that quantitative polymerase chain reaction methods (qPCR) show high inter-lab variabilities which makes it substantially difficult to compare the dosing of different vectors used in different studies. The comparability problem could be solved by the availability of suitable standard material for the analytical characterization of AAV gene therapy products. At the moment there is Reference Standard Material (RSM) for the AAV serotypes AAV2 and AAV8, however, there is no comparable material for the remaining AAV serotypes. Though each lab has its own internal reference material, the comparability between different labs remains difficult due to the variability of available quantification methods and the lack of RSM.   

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The Challenges of AAV-Based Gene Therapy

Dr. Hüseyin Besir (Director R&D) and Dr. Dana Holzinger (Head of Product Management) discuss the challenges faced with AAV-based Gene Therapy

Adeno-associated virus (AAV) has become one of the most popular vectors for gene delivery in various in vivo gene therapy applications. However, a lack of suitable and widespread reference material in gene therapy is just one of the many obstacles facing laboratories around the world attempting to find treatments for genetic diseases. Download the article and continue reading the full article from the European Biopharmaceutical Review.

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AAV VP Protein Standards

The progress into clinical trials as well as the increasing demand for AAV gene therapy vectors in the field in general leads to an increasing need of high yields, high purity and high quality AAV particles. Therefore, it gets more and more important to ensure a comprehensive monitoring during the vector manufacturing process. Monitoring the VP1, VP2 and VP3 expression levels as well as the expression ratios of the AAV capsid proteins allow to optimize the yield and the potency of the production at an early time point.

The AAV2 VP protein standard can be used to create mixtures of VP1, VP2 and VP3 with a precise molar ratio, e.g. 1:1:10 for comparison with the protein composition of your sample by protein detection methods like western blot. All three AAV2 proteins are available as individual proteins (10 µg each) and can be used to prepare specific VP protein mixtures as needed. 

AAV2 VP Protein Standards (100 µg/ml)

1.19 µM
1.45 µM
1.61 µM

AAV ELISA Controls

The AAV ELISA controls offered by PROGEN can be used as positive controls with the corresponding AAV ELISAs. They consist of fully assembled, empty AAV particles and have been characterized according to our internally established reference standrad material. The reference standard material contains a GFP reporter gene and has been characterized by qPCR & ddPCR (DNA quantification) as well as electron microscopy including negative stain EM and cryo EM (ratio of full to empty capsids).

Each lot of AAV ELISA controls within a specific serotype gets carefully calibrated and might differ in concentration due to variabilities generally observed for AAV vector productions. Please find the concentration range for each serotype below.

Available AAV ELISA Controls including concentration range:

1.2E+09 - 2.0E+09
1.7E+09 - 2.8E+09
1.1E+09 - 1.9E+09
 7.9E+09 - 1.3E+10

2.0E+09 - 3.3E+09
3.8E+08 - 6.3E+08
9.0E+08 - 1.5E+09

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