AAV Packaging Workflow

I. Overview of rAAV Packaging

Recombinant adeno-associated virus (rAAV) production is primarily based on triple-plasmid co-transfection of HEK293T cells, consisting of:

• Transfer plasmid (pAAV-GOI/shRNA), containing the gene of interest flanked by ITRs

• Packaging plasmid (pAAV-RC), providing Rep and Cap proteins

• Helper plasmid (pHelper), supplying adenoviral helper functions

During packaging, HEK293T cells provide AAV structural proteins (Cap) and functional proteins (Rep) in trans. With the assistance of the helper plasmid, the ITR-flanked target DNA is efficiently packaged into rAAV viral particles.

Figure 1. rAAV production workflow

1. rAAV Vector Construction

• Vector design and cloning
  Appropriate rAAV expression vectors are selected based on the experimental objective and target cell type. The gene of interest is typically cloned into the rAAV transfer plasmid using standard restriction enzyme digestion–ligation–transformation–sequencing workflows. Vector integrity and expression functionality are then validated.

• Plasmid DNA preparation
  To obtain high-quality, high-concentration, low-endotoxin plasmid DNA, plasmids are purified using column-based extraction methods in combination with endotoxin removal reagents.

Figure 2. Construction workflow of the rAAV transfer plasmid

2. rAAV Packaging Procedure (Adherent HEK293T Cells)

(1) Cell preparation

Cryopreserved HEK293T cells are retrieved from liquid nitrogen and rapidly thawed in a 37 °C water bath. After centrifugation, fresh culture medium is added and cells are cultured at 37 °C with 5% CO₂. Cells are passaged every 2–3 days. Once normal growth is observed, cells are seeded into 10-cm culture dishes and allowed to adhere.

(2) Plasmid transfection

When cell confluency reaches approximately 80–90%, transfection is performed. The three plasmids are mixed at a defined ratio, and transfection is carried out using a total volume of 1,000 μL per dish, followed by incubation under standard culture conditions.

(3) Post-transfection culture

Twenty-four hours post-transfection, transfection efficiency is evaluated by microscopy. In general, transfection efficiency should exceed 80%, as lower efficiency can significantly reduce viral yield.

(4) Virus harvest and clarification

At 72 hours post-transfection, viral supernatant is collected. The supernatant is clarified by low-temperature centrifugation to remove cell debris, followed by treatment with nuclease at 37 °C to degrade free nucleic acids. PEG8000/NaCl solution is then added, and viral particles are precipitated overnight.

(5) Virus purification and concentration

The precipitated virus is resuspended in PBS and subjected to density gradient ultracentrifugation. After centrifugation, the virus-containing fraction is collected and dialyzed overnight at 4 °C using dialysis tubing. The following day, the dialysate is filtered through a 0.22 μm membrane and further concentrated and buffer-exchanged using centrifugal concentrators to achieve purification and concentration.

II. rAAV Quality Control

Quality assessment of rAAV includes titer determination, purity analysis, sterility testing, mycoplasma detection, and endotoxin testing.

(1) Viral genome titer determination

Method: Each intact rAAV particle contains a single copy of the viral genome. Viral samples are subjected to acid–base lysis to release genomic DNA, followed by quantitative PCR (qPCR) using sequence-specific primers. The number of viral genomes (VG) is calculated and expressed as VG/mL.

QC criterion:
≥ 1.0 × 10¹² VG/mL

(2) Purity analysis

Method: Viral purity is assessed by SDS–PAGE, followed by silver staining.

QC criterion:
Only three bands corresponding to VP1, VP2, and VP3 should be present, with an approximate intensity ratio of 1 : 1 : 10, and no significant contaminating bands.

(3) Sterility testing

Method: Viral samples are inoculated into culture media suitable for bacterial and fungal growth and incubated at 37 °C for 7 days.

QC criterion:
Culture media must remain clear and transparent, with no bacterial or fungal growth.

(4) Mycoplasma detection

Method: Primers targeting conserved regions of mycoplasma 16S rRNA genes are used for PCR amplification, followed by gel electrophoresis and/or qPCR analysis.

QC criterion:
No amplification band detected on agarose gel.

(5) Endotoxin testing

Method: Endotoxin levels are measured according to the instructions of the Limulus Amebocyte Lysate (LAL) assay kit (quantitative chromogenic tube method).

QC criterion:
< 10 EU/mL