The Challenges of AAV-Based Gene Therapy

Background

Adeno-associated virus (AAV) has become one of the most popular vectors for gene delivery in various in vivo gene therapy applications. Consequently, the gene therapy community is experiencing an exponential increase in the number of clinical trials, most of which are being conducted in the USA. 

This is creating an increase in the number of companies specializing in the production of gene therapies for the treatment of genetic disorders. However, a lack of suitable and widespread reference material in gene therapy is just one of the many obstacles facing laboratories and regulatory authorities around the world attempting to find treatments for genetic diseases.

Finding the right dose

AAV-based gene therapies reach from small dose applications to high doses depending on the target organ or tissue. Yet, 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. AAV-based gene therapies are limited to a single application, so choosing the right dose is a critical step.

Comparability

The dose of AAV gene therapies is usually defined by the copy number of viral genomes per kilogram (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 dose 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, but 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.

Further challenges

Besides the contribution of pre-existing conditions, principal issues in transferability of data from animal experiments, as well as comparability of meaningful titers, can pose additional difficulties for the evaluation of the safety of a specific product.

For an deeper divev into the challenges of AAV-Based Gene Therapy read the following article as featured in the European Biopharmaceutical Review.

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


 

AAV VP Protein
Standards


The progress into clinical trials as well as the increasing demand for AAV gene therapy vectors in the field leads to an increasing need of high yields, high purity and high quality AAV particles. Therefore, it’s becoming even 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 allows yield optimization and the monitoring of potency production early on. 


AAV2 VP Protein Standards (100 µg/ml)

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 using 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.

Product features include:

  • Reference standard for ratio analysis
  • Precise molar concentration 
  • Suitable for a variety of applications including WB, dot blot, CE, SDS PAGE 
  • Available as a recombinant AAV2 VP1-3 capsid protein set


VP1
VP2
VP3
1.19 µM
1.45 µM
1.61 µM

AAV Empty Capsids

PROGEN offers comprehensively characterized AAV empty capsids which can be used as reference material in a variety of applications. These are available for AAV1, AAV2, AAV5, AAV6, AAV8 and AAV9.

Product features include:

  • Free from additives and stabilizing proteins
  • Liquid formulation
  • Titer above 5.0E+12 viral particles/ml
  • Quality controlled - according to PROGEN´s well-established QC standards
  • Suitable for a variety of applications

The high titer AVV empty capsids are provided in a liquid formulation in PBS with 0.014% Tween, 1 mM MgCl2 and 2.5 mM KCl. This formulation and the high titers of more than 5.0E+12 viral particles/ml allow the empty capsids to be used as a reference standard in different applications e.g. dot blot, western blot as well as ELISA.

Quality assured

PROGEN follows a well-established QC protocol for the characterization of the AAV empty capsid material, to ensure a comprehensive characterization and a reliable titer determination for each production. The QC protocol includes the analysis of sample purity by SDS PAGE, analysis of the filling grade by AUC as well as the titer determination using our PROGEN´s carefully calibrated ELISA kits and internal reference material/international RSM for AAV2 and AAV8. Due to the extensive QC and the reliable characterization of the AAV empty capsids, they are suitable for use as reference standard to ensure the validity of several assays.

For more information regarding PROGEN's reference material download our poster „Developing Reliable AAV Standards for ELISA“ or take a look at the Technical Performance Data of PROGEN´s serotype-specific AAV ELISAs.

View All Empty Capsids

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 standard material or the international RSM for AAV2 and AAV8. 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).

Product features include:

  • Fully assembled, empty AAV particles
  • Reliable positive control for ELISA
  • Quality controlled - according to PROGEN´s well-established QC standards



Available AAV ELISA Controls including concentration range:

Each lot of AAV ELISA controls within a specific serotype is carefully calibrated. They might differ in concentration due to variabilities generally observed for AAV vector productions.



AAV1
AAV2
AAV3
AAV5
1.2E+09 - 2.0E+09
1.7E+09 - 2.8E+09
1.1E+09 - 1.9E+09
 7.9E+09 - 1.3E+10


AAV6
AAV8
AAV9
2.0E+09 - 3.3E+09
3.8E+08 - 6.3E+08
9.0E+08 - 1.5E+09

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