Adapting A Replication Competent Adeno-Associated Virus (rcAAV) Assay For Commercial Manufacturing

By Oxana M. Tsygankova, Ph.D., Leann Walsh, Lana Sweet, and Dana Cipriano, Department of Virology, Testing & Analytical Services; and Brian Tomkowicz, Ph.D., Viral Vector Research and Development, SK pharmteco – US

The triple transfection method for generating adeno-associated virus (AAV) particles is a key technique in recombinant AAV (rAAV) production, vital for gene therapy. This method involves co-transfecting three plasmids into human embryonic kidney 293 (HEK293) cells: the Rep/Cap plasmid, the adenovirus helper plasmid, and the AAV genome plasmid. The Rep/Cap plasmid provides the essential replication and capsid proteins, determining the AAV serotype and tissue tropism. The adenovirus helper plasmid supplies necessary helper genes to support AAV replication and packaging. The AAV genome plasmid contains the transgene flanked by inverted terminal repeats (ITRs), which are crucial for the stability, replication, and packaging of the AAV genome.

In gene therapy, replacing the native rep and cap genes with separate plasmids allows for the creation of rAAVs with customizable properties. This triple transfection method is highly efficient and adaptable, enabling the rapid production of various AAV serotypes by altering the cap gene. Advances in serum-free suspension cell lines have made it possible to scale up this process for larger production volumes, although optimization is necessary to maintain yield and purity.

AAV genome packaging involves a precise interaction between the Rep protein, ITRs, and rep-binding elements (RBEs). ITRs, located at both ends of the AAV genome, are essential for replication, packaging, and integration, serving as binding sites for Rep proteins and host factors. RBEs within the ITRs are specifically recognized and bound by Rep proteins, facilitating key steps in the AAV life cycle, including genome replication, capsid assembly, and integration into the host genome.

HEK293 cells, with their stably integrated adenovirus E1A proteins, are particularly suited for rAAV production due to their ability to support robust AAV vector generation. The interaction between E1A and the P5 promoter of the AAV genome, which controls rep gene expression, is crucial. E1A proteins activate the P5 promoter, enabling AAV production, while Rep proteins regulate their own expression through feedback mechanisms at the P5 promoter.

Understanding these interactions—between Rep proteins, RBEs, and the P5 promoter—allows for improved AAV vector design and gene therapy applications. By manipulating these elements, researchers can enhance vector yield, specificity, and safety, reducing risks such as insertional mutagenesis. Ongoing research continues to explore these complex interactions to optimize AAV-based gene therapies, aiming to enhance their efficacy and safety.