Applications and Storage of Adeno-Associated Virus (AAV)

Adeno-associated virus (AAV) is a non-pathogenic, single-stranded DNA virus belonging to the Parvoviridae family, first discovered as a contaminant in adenovirus preparations, hence its name. AAV consists of a protein capsid enclosing a single-stranded DNA genome containing genetic information. AAV exhibits excellent biosafety, with minimal pathogenicity to humans, making it widely used in gene therapy, gene delivery, and gene editing fields. As a dependovirus, wild-type AAV requires helper viruses (like adenovirus or herpesvirus) for replication, but recombinant AAV (rAAV) vectors used in applications are replication-defective for enhanced safety.

Main Application Areas of AAV:

• Gene Therapy: AAV is extensively applied in gene therapy to deliver therapeutic genes into patients’ cells. This provides new possibilities for treating genetic diseases and other refractory conditions, such as spinal muscular atrophy (SMA), hemophilia, and inherited retinal disorders. Approved therapies like Luxturna (for Leber congenital amaurosis) and Zolgensma (for SMA) demonstrate AAV’s clinical success, leveraging its low immunogenicity and ability for long-term transgene expression.
• Gene Delivery: AAV can introduce exogenous genes into in vitro cultured cells for studying gene function, regulation, and related mechanisms. It serves as a common tool in scientific research, particularly for stable expression in hard-to-transfect cells like primary neurons or stem cells.
• Immunotherapy: AAV can be used in immunotherapy by introducing specific immune-related genes to enhance the body’s immunity, combating certain types of cancer or infections. For example, AAV vectors are explored in CAR-T cell engineering or vaccine development to elicit targeted immune responses.
• Gene Editing: AAV serves as a delivery vehicle for gene editing tools, such as the CRISPR-Cas9 system, enabling precise genome modifications in cells. Its non-integrating nature (primarily episomal persistence) reduces off-target risks compared to integrating vectors, though integration can occur at low frequencies.

AAV Storage Methods:

To ensure long-term preservation and stability of AAV, the following storage methods can be adopted:

• Low-Temperature Storage: AAV viruses should be stored in an ultra-low temperature freezer at -80°C or lower to slow down degradation processes.
• Aliquoting: Divide AAV viruses into small-volume, single-use cryotubes or vials to avoid repeated freeze-thaw cycles, which reduce damage to the virus.
• Addition of Protectants: During storage, consider adding cryoprotectants such as glycerol (typically 10-20%) or sucrose to help prevent freeze-thaw damage.
• Avoid Light Exposure: Store cryotubes or vials under light-protected conditions to prevent adverse effects from ultraviolet light on the virus.
• Detailed Labeling: Mark detailed information on cryotubes or vials, including AAV serotype, titer (e.g., VG/mL), vector type, and production date, for accurate identification during use.

Through scientifically sound storage methods, AAV’s long-term efficacy can be ensured, allowing it to perform stably and reliably in applications such as gene therapy, gene delivery, and gene editing. For added stability, AAV stocks should undergo minimal freeze-thaw cycles (ideally <3-5), and post-thaw titer verification via qPCR or functional assays is recommended to confirm integrity. High-purity AAV preparations (e.g., <30% empty capsids) maintain better stability during storage.