AAV Packaging Processes: Roles and Techniques

Adeno-associated virus (AAV) vectors are widely used in gene therapy for their efficiency in delivering genetic material with minimal immune response. Packaging these vectors is essential to producing high-quality viral particles for research and clinical applications.

Producing AAV vectors involves specific genes, plasmids, and cell lines to assemble and package the viral genome. Each component plays a distinct role in generating a functional vector for therapeutic use.

Structure Of AAV Vectors

AAV vectors are engineered viral particles designed to deliver genetic material into target cells. They consist of a non-enveloped icosahedral capsid, approximately 25 nanometers in diameter, enclosing a single-stranded DNA genome. Unlike wild-type AAV, which requires a helper virus for replication, recombinant AAV (rAAV) vectors lack viral genes responsible for replication, making them replication-deficient and safer for therapeutic applications. This modification allows for the insertion of a therapeutic transgene while maintaining structural integrity for efficient cellular entry and gene expression.

The AAV genome, about 4.7 kilobases long, consists of three primary components: the transgene cassette, which carries the therapeutic gene; the promoter, which regulates gene expression; and the inverted terminal repeats (ITRs), the only viral sequences retained from the wild-type genome. These ITRs facilitate genome replication and packaging by forming secondary structures that aid viral DNA processing. Removing the rep and cap genes from the vector genome prevents autonomous replication, requiring external helper elements during manufacturing.

The capsid, composed of viral proteins VP1, VP2, and VP3 in a 1:1:10 ratio, determines the vector’s ability to target specific cell types. Different AAV serotypes, such as AAV2, AAV5, and AAV9, exhibit distinct capsid structures that influence tissue affinity. For example, AAV9 crosses the blood-brain barrier, making it ideal for neurological disorders, while AAV8 exhibits strong liver tropism, enhancing its suitability for hepatic gene therapies. The capsid also mediates cellular entry by interacting with surface receptors and co-receptors, triggering endocytosis. Once inside the cell, the vector undergoes endosomal processing and nuclear transport, where its single-stranded genome is converted into a double-stranded form for transcriptional activation.

Roles Of Rep And Cap Genes

Recombinant AAV vector production relies on the rep and cap genes, derived from the wild-type AAV genome. These genes encode essential proteins for genome replication and capsid formation. By providing them in trans during production, rAAV remains replication-deficient while still being efficiently packaged, improving biosafety and enabling controlled vector manufacturing.

The rep gene encodes four overlapping proteins—Rep78, Rep68, Rep52, and Rep40—produced through alternative splicing and differential promoter usage. Rep78 and Rep68 facilitate genome replication by recognizing terminal sequences and initiating replication, while Rep52 and Rep40 assist in packaging by unwinding and transporting the replicated single-stranded DNA into capsids. Without Rep proteins, genome amplification and encapsidation cannot occur.

The cap gene encodes VP1, VP2, and VP3, which form the icosahedral capsid in a tightly regulated ratio. VP3 provides structural stability, while VP1 and VP2 contribute to receptor binding and intracellular trafficking. The cap gene also determines serotype specificity, as variations in capsid proteins influence tropism and cellular entry. This serotype diversity allows researchers to tailor vectors for specific therapeutic applications.

Helper Plasmids And Functions

Helper plasmids provide the necessary elements for AAV replication and packaging, compensating for the missing viral genes in the rAAV genome. By supplying replication and structural components in trans, these plasmids enable scalable and biosafe vector production.

AAV production typically requires at least two helper plasmids: one encoding the rep and cap genes and another supplying adenoviral helper functions. The rep-cap plasmid provides replication and capsid proteins, while the adenoviral helper plasmid contains genes such as E2A, E4, and VA RNA, which stimulate AAV replication. These adenoviral elements activate the host cell’s DNA replication machinery, mimicking the role of a wild-type helper virus without introducing a fully functional adenovirus. This system prevents contamination with replication-competent viruses while maintaining high vector yields.

Optimizing plasmid design enhances vector production. Codon optimization of rep and cap genes improves protein expression, while stronger promoters like cytomegalovirus (CMV) or chicken β-actin (CBA) enhance transcription. Modifications to the adenoviral helper plasmid, such as removing unnecessary sequences, can reduce cytotoxicity and prolong cell viability, leading to higher vector titers and improved consistency across manufacturing batches.

Packaging Cell Lines

Recombinant AAV vector production relies on specialized packaging cell lines that support genome replication and capsid assembly. These cell lines influence vector yield, purity, and quality, making optimization a key focus in AAV bioprocessing.

HEK293 cells, derived from human embryonic kidney cells, are the most widely used platform for AAV production due to their high transfection efficiency and protein expression. Their integrated adenoviral E1 gene complements the adenoviral helper plasmid, enhancing AAV replication. Variants like HEK293T and HEK293FT offer additional advantages, such as faster growth rates and improved transfection efficiency.

Suspension-adapted HEK293 cells, grown in serum-free media, enable large-scale vector production in bioreactors. These cultures eliminate the need for adherent cell substrates, reducing labor-intensive processes and allowing for higher-density cultures that maximize vector yield. The shift to suspension-adapted HEK293 cells has been instrumental in meeting the growing demand for clinical-grade AAV vectors.

Steps In The Packaging Process

AAV packaging involves transfection, vector replication and assembly, and viral particle harvest. Each step is optimized to maximize yield while maintaining viral particle integrity.

The process begins with the co-transfection of HEK293 cells with three plasmids: the rAAV genome plasmid, the rep-cap plasmid, and the adenoviral helper plasmid. Chemical reagents like polyethyleneimine (PEI) or calcium phosphate facilitate plasmid introduction into cells. Once inside, the rep and cap genes drive genome replication and capsid formation, while adenoviral helper functions enhance replication. Over 48 to 72 hours, cells produce and package AAV genomes into capsids, accumulating viral particles in the cytoplasm.

Following vector assembly, viral particles must be released from the cells. This is achieved using lysis methods such as freeze-thaw cycles, enzymatic digestion, or sonication. The resulting crude lysate contains fully packaged AAV particles, empty capsids, cellular debris, and residual plasmid DNA. Clarification removes large contaminants before purification, which is essential to eliminate empty capsids and impurities that could impact vector potency.

Purification And Titer Analysis

Purification removes contaminants and enriches fully packaged vectors, enhancing potency and ensuring regulatory compliance for clinical applications.

Density gradient ultracentrifugation, using iodixanol or cesium chloride, separates full capsids from empty or partially packaged ones based on buoyant density. While effective, ultracentrifugation is labor-intensive and difficult to scale, leading many manufacturers to adopt chromatography-based methods. Affinity chromatography, using ligands like heparin or AAV-specific antibodies, provides high selectivity for intact viral particles. Ion-exchange and size-exclusion chromatography further refine purity and concentration, facilitating large-scale production with batch-to-batch consistency.

Titer analysis quantifies the final vector preparation. Quantitative PCR (qPCR) or droplet digital PCR (ddPCR) measure genome copies, while enzyme-linked immunosorbent assays (ELISA) estimate total capsid content. Functional assays, such as transduction-based measurements, assess infectivity. Accurate titer assessment is critical for dosing precision in therapeutic applications, as variations in vector potency can affect treatment outcomes.