Adeno-associated virus (AAV) Packaging: A Comprehensive Exploration of its Packaging, Mechanisms, and Diverse Applications

During the AAV packaging process, the packaging plasmid encodes the target gene along with two inverted terminal repeat sequences (ITR), which play a crucial role in virus replication and packaging. The auxiliary plasmid contains the cap gene (encoding the viral capsid protein) and the rep gene (involved in virus replication), as well as the adenovirus Helper plasmid. These three plasmids are co-transfected into 293T cells, initiating AAV virus replication and packaging. The production and quality control of AAV from Paizhen Biotech follows internationally recognized standard procedures, utilizing a three-plasmid system for packaging in 293T cells. The obtained virus particles are purified by ultracentrifugation and titrated by qPCR for viral genome copies. Additionally, upon request, protein staining can be provided to assess the integrity of the capsid protein and endotoxin content. Typically, the titer of rAAV ranges from 10^12 to 10^13 VG/ml, which is sufficient to meet the requirements of various experiments.

Titer Quantification: Quantitative PCR detects the copy number of exogenous DNA in the viral genome.

Principle of Titration: The genome of adeno-associated virus is single-stranded DNA, and the copy number of exogenous DNA represents the copy number of the viral genome.

Purified AAV viral vectors can be used to infect cells. During cell infection, AAV binds to specific receptors on the cell surface, activating intracellular signaling pathways, thereby triggering AAV entry into the cell through receptor-mediated endocytosis. With the assistance of cellular organelles such as the endosome and Golgi apparatus, AAV enters the cell nucleus. Subsequently, the virus is uncoated, and its single-stranded DNA needs to be replicated into double-stranded DNA before expressing the target gene.

rAAV Mechanism of Action

1. High safety and low immunogenicity: AAV is a replication-defective DNA virus with no autonomous replication capability. Wild-type AAV relies on the rep gene for low-frequency site-specific integration, whereas rAAV does not integrate. There have been no reports of human or mammalian diseases caused by AAV, making it one of the safest viral vectors approved by the FDA for gene therapy.
2. Broad host cell range: AAV has a wide host range, infecting both dividing and non-dividing cells.
3. Strong diffusion ability: With a diameter of approximately 20-26 nm and high titer, AAV exhibits excellent diffusion ability. AAV-PHP.eB and AAV9, in particular, have the ability to cross the blood-brain barrier, making them widely used in neuroscience.
4. Long-term gene expression in vivo: AAV can maintain long-term gene transcriptional expression ability. Peak expression is generally reached within 3 weeks, followed by sustained high expression for over 5 months.
5. Diverse serotypes: AAV has numerous serotypes, and new serotypes are continuously being mutated and screened based on different experimental designs (AAV1-13, AAV2/1, 2/2, 2/5, 2/6, 2/8, 2/9, DJ; retro, PHP.eB…).

Comparison of Biological Characteristics of Different Viruses

Based on the above characteristics, AAV is considered an efficient and safe tool for both in vitro and in vivo gene transfer. Especially in whole-level studies, compared to other commonly used viral vector carriers, AAV exhibits mild and long-lasting expression capabilities during the infection process, making it a powerful tool for genetic manipulation.

Currently, the total number of registered AAV serotypes exceeds 196, with 13 different serotypes (AAV1-AAV13) found in primates. Among them, AAV2 and AAV3 originate from humans. AAV2, being the earliest cloned virus, is the most thoroughly researched and widely applied viral vector to date. With ongoing research, scientists have discovered that different serotypes of AAV can undergo hybridization, resulting in hybrid AAV with characteristics of both parents. Consequently, AAV subtypes emerge.

The commonly used rAAV in current research is produced by combining the AAV2 genome with different capsid proteins, resulting in hybrid viral vectors labeled as rAAV2/N (N represents different capsid serotypes). Recombinant viruses retain the stable expression and gene integration capabilities of AAV2 while gaining tissue infectivity specificity for different serotypes (specific structural sites on the surface of capsids of different serotypes determine the specificity of their respective receptors), exhibiting certain organ targeting specificity.

Tissue Tropism of Different AAV Serotypes

PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.

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