An Approach for Enriching the Production of DNA Cargo Loaded AAV5 Capsids in Cells
This technology provides an improved method for producing recombinant adeno-associated viruses (rAAVs) by introducing four genetic constructs into host cells, each encoding component essential for efficient rAAV packaging. By optimizing the timing of transfection and expression, this system enhances the likelihood that capsids are correctly assembled and filled with therapeutic DNA. This results in a substantially higher proportion of functional, genome-containing AAV particles. By improving coordination between genome replication and capsid assembly, this approach overcomes limitations of current production methods, such as low yields and the formation of empty or defective capsids.
Researchers
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methods and compositions for enriching the production of dna cargo loaded recombinant adeno-associated virus capsids
Patent Cooperation Treaty | Pending
Technology
This technology uses a two-stage transfection strategy to increase the proportion of rAAV capsids that are properly filled with therapeutic DNA. In the first stage, host cells are transiently transfected with three constructs: one encoding adenoviral helper genes (E2A, E4Orf6, and VA RNA), a second encoding Rep proteins, and a third carrying the gene of interest. In the second stage, a fourth construct is introduced containing additional Rep and capsid (Cap) genes. This fourth construct drives AAV genome replication and capsid assembly. The transfected cells are then maintained under optimized conditions to support efficient rAAV packaging before purification.
Problem Addressed
A major challenge in rAAV production is the low yield of functional viral particles that are correctly filled with therapeutic genetic material. This inefficiency not only limits the effectiveness of gene therapy, but also complicates purification, increasing costs and causing product loss. Separating intact vectors from empty or defective particles is not only time-consuming, but also technically demanding. Furthermore, temporal mismatches between capsid assembly and viral genome replication often result in empty or partially filled capsids.
Advantages
- Higher yield of functional, genome-filled rAAV particles.
- Simplified purification due to fewer empty or defective capsids.
- Scalable and flexible process using modular constructs.
- Improved vector quality due to better control of Rep/Cap expression by staggering transfections.
Publications
Nguyen, T. N. T., D. Park, C. T. Canova, J. Sangerman, P. Srinivasan, R. W. Ou, P. W. Barone, et al. “Perfusion-Based Production of rAAV via an Intensified Transient Transfection Process.” Biotechnology and Bioengineering 122, no. 6 (June 2025): 1424–1440. https://doi.org/10.1002/bit.28967.
Srinivasan, P., C. T. Canova, S. Sha, T. N. T. Nguyen, J. Joseph, J. Sangerman, A. J. Maloney, et al. “Multidose Transient Transfection of Human Embryonic Kidney 293 Cells Modulates Recombinant Adeno-Associated Virus2/5 Rep Protein Expression and Influences the Enrichment Fraction of Filled Capsids.” Biotechnology and Bioengineering 121, no. 12 (December 2024): 3694–3714. https://doi.org/10.1002/bit.28828.
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