AAV Gene Therapy Explained: The Critical Role of Accurate QC Testing

For gene therapy, viral vectors are commonly used to deliver therapeutic genes into patients' target cells with the aim of treating genetic diseases. Adeno-associated virus (AAV) vectors are among the most prominent viral vectors for in vitro gene therapy, and their investigation and development have gained increasing interest.

In this article you will learn about the role of AAV vectors in gene therapy and why accurate AAV vector titer testing is crucial for safe and efficacious gene therapy products.

What is Gene Therapy?

Gene therapy represents one of the most ground-breaking advances in modern medicine, employing a range of strategies to modify gene expression within target cells. Instead of targeting symptoms, gene therapy uses viral or non-viral vehicles to deliver therapeutic genes to cells or tissues to modify or exchange defective genes. This strategy allows genetic disorders to be treated at their root cause and has shown promising results in treating inherited disorders, certain cancers, and even rare diseases that previously had no therapeutic options. Over the past 10 years, several gene therapies have received regulatory approval in both the United States (FDA) and Europe (EMA), marking a turning point for genetic medicine.

Why AAV Vectors are so Important?

One of the most effective vehicles for delivering therapeutic genes is the adeno-associated virus (AAV).
AAV is a non-enveloped, single-stranded DNA virus that is engineered as a vector to carry therapeutic DNA into human cells.

AAV vectors have emerged as the preferred choice because of their long-term gene expression, broad tissue tropism, good safety profile, and versatile manufacturing processes.
Importantly, AAV is non-pathogenic, and rarely integrates into the host genome. ^[1]

Key Benefits of AAV Vectors

 
With multiple naturally occurring serotypes and engineered variants available, AAVs can be tailored for muscle, liver, central nervous system, or ocular delivery, making them versatile across a wide range of therapies. Up to now, scientists have identified 13 different human AAV serotypes (AAV1-13) that are characterized by different capsid proteins. AAV2 is the most thoroughly investigated serotype.

Examples of Applications

In the last 10 years, several AAV-based gene therapies have been approved in both the United States and Europe, highlighting the rapid progress of this field. Products such as Luxturna® for inherited retinal dystrophy, Zolgensma® for spinal muscular atrophy, and Hemgenix® for haemophilia B are already transforming patient care.

These ground-breaking therapies demonstrate how AAV vectors can be harnessed to restore vision, replace missing proteins in blood clotting disorders, or deliver life-saving genes to neurons — offering cures for diseases that were once untreatable.

Manufacturing Process of AAV Vectors

The production of AAV vectors involves several key steps:

1. Cell Culture & Transfection
   Producer cell lines (commonly HEK-293 cells) are transfected with plasmids encoding the therapeutic gene, helper functions, and capsid proteins.
2. AAV Assembly & Packaging
   Inside the host cells, the therapeutic DNA is packaged into AAV capsids. This creates functional viral particles capable of gene delivery.
3. Harvesting & Purification
   AAVs are harvested from the cells and purified using chromatography or density gradient ultracentrifugation. This removes host cell proteins, DNA, and other contaminants.
4. Formulation
   The purified AAV vector is formulated into the final drug product, ready for preclinical and clinical use.

Throughout this process, quality control (QC) testing plays a vital role to confirm that the vector is pure, potent, and safe.

Quality Control of AAV gene therapy products

To ensure product quality and safety it is essential to establish critical quality attributes (CQAs). One CQA is the AAV vector genome titer. Specificity of AAV vectors as well as the concentration of the vector titer must be determined throughout the production process and for final release testing. In addition, rigorous testing for potential mycoplasma contamination and traces of residual host cell DNA is essential to ensure the safety of the final product.

Required Quality Control Tests

The central Role of AAV Vector Titer Testing

Among all required QC tests, AAV vector titer testing is particularly crucial because it directly influences the dose accuracy of a gene therapy product. Inaccurate titer measurements can result in underdosing or overdosing - reducing therapeutic efficacy or raising safety concerns.

Traditional methods, such as qPCR, can be susceptible to variability due to differences in sample preparation, and the need for a standard curve, which relies on error-prone dilution series. In contrast, digital PCR offers a powerful solution to these challenges by enabling absolute quantification without the need for a standard curve. This level of accuracy is particularly valuable in the context of AAV vector titer testing, where an accurate result is critical for dose determination and regulatory compliance.

How MiQuant® AAV Sets a New Standard

Our ready-to-use MiQuant® AAV kits provide fast and accurate quantification of AAV vector genome titers, supporting both in-process control and final QC release testing.
Tailored kit versions are available for the specific detection of AAV ITR 2/6 (also covering ITR 1, 3, and 7) as well as ITR 5.
They are based on digital PCR (dPCR) and compatible with a wide range of dPCR/ddPCR™ platforms.
 

Ultra-Precise AAV Vector Titer Quantification

The integrated TruTiter™ technology (powered by Promega) ensures that only the DNA packaged within intact AAV capsids is amplified, while free DNA and DNA from broken capsids remain masked and undetectable. This enables highly precise measurement of AAV vector genome titer and eliminates the need for a DNase treatment step. Unlike DNase, which cannot access DNA enclosed in damaged capsids, TruTiter™ provides superior accuracy and reliability in vector quantification.

^[1] https://www.nature.com/articles/s41392-024-01780-w