Advances in genetics and bioengineering have enabled the development of gene therapy to treat diseases caused by recessive gene disorders, acquired genetic diseases, and some viral infections. Technologies for the manufacture and analysis of gene therapy products including adeno-associated virus (AAV) vectors are evolving with continued efforts to be fully optimized.
As you seek to understand more about AAV characterization, read the Q&A below to accelerate your learning:
What are adeno-associated virus (AAV) vectors?
Adeno-associated virus (AAV) vectors are the leading platform for gene delivery for the treatment of a variety of human diseases. AAVs consist of an icosahedral protein capsid built by 60 copies of three proteins (VP1, VP2, VP3), resulting in an average weight of approximately 3.5 MDa. The protein capsid contains a single-stranded DNA genome, adapted for therapeutic purposes.
Why are AAV vectors important?
Recent advances in developing clinically desirable AAV capsids, optimizing genome designs and harnessing revolutionary biotechnologies have contributed substantially to the growth of the gene therapy field. Continued study of AAV biology and increased understanding of the associated therapeutic challenges and limitations will build the foundation for future clinical success.
Why is AAV characterization challenging?
Gene therapy using viral vectors, such as AAV, is a rapidly evolving area of biopharmaceutical research with multiple clinical studies showing promising results in treating severe disease. However, despite this great advancement, manufacturing as well as downstream processing and analytical characterization of AAVs remains challenging mainly due to sample yields, long turnaround times for established methodologies, and serotype complexity.
How do AAV vectors support gene therapy?
Gene therapy involves either supplying or suppressing a specific gene that’s either lacking in a patient or causing the disease, respectively. In both cases the treatment works through the delivery of therapeutic DNA to target cells. The delivery strategy is based on both viral and non-viral vectors, with the most common being AAV vectors. Hence, analytical technologies that can monitor the production and correct assembly of viral capsids, as well as encapsulation of the gene used to treat the disease, have become of pivotal importance for the further development of this promising area.
What’s the purpose of AAV product characterization?
Characterization of AAVs includes a number of critical parameters that need to be assessed during production, such as:
- Vial protein ratio to assure transduction efficiency and correct genome delivery and release
- Vector yield to ensure enough of the therapeutic gene is administered
- Titer of the viral capsids that will be used in the subsequent production processes
- Amount of residual empty capsids following downstream processing
Why is the titer of viral capsids important?
There are several cellular expression systems for recombinant AAV production, with the most common ones being mammalian HEK293 cells or SF9 insect cells. The inherent complexity of recombinant protein manufacturing requires detailed monitoring of various product quality attributes (PQAs), such as correct capsid assembly and packaging but also the assessment of obtained product titers. Knowing the exact titer is not only important to monitor production efficiency in general, but also to determine the correct dosage for subsequent treatment. You can learn more in this Knowledgebase post: Gene therapy: Using size exclusion chromatography to evaluate AAV product titers.
Why is measuring empty capsids important?
In recent years, recombinant AAV production has been steadily optimized to yield high genome packaging efficiency; but empty capsids often remain an undesired byproduct during manufacturing. Additionally, some evidence shows immune reactions caused by empty capsids. Therefore, it’s important to know the precise amount of correctly filled AAV capsids in order to determine the correct dosage required for treatment.
How do labs characterize AAV capsids for genome packaging efficiency?
There are several techniques available to measure the amount of empty and full capsids. The most common are analytical ultracentrifugation (AUC) and, more recently introduced, anion-exchange chromatography (AEX) and capillary electrophoresis (CE). Yet, those methods rely on absorbance-based detection which can be problematic. One limitation is a lack of sensitivity, which is of significance when working with expensive and low-concentrated samples. More importantly, absorbance-based methods do not allow for a definite identification of full and empty capsid species, which can be successfully circumvented by employing mass spectrometry (MS).
Even still, native MS analysis of intact viral capsids can be challenging due to their high molecular weight and complexity. However, one recent study presented a native mass spectrometry-based approach to assess the ratio of empty to full AAV capsids without the need for excessive sample preparation. This work demonstrated the potential of native mass spectrometry for the characterization of viral particles.
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