As the field of gene therapy continues to evolve, the demand for reliable and efficient AAV packaging services has never been greater. By combining cutting-edge scientific expertise with personalized service, Protheragen is committed to providing AAV packaging services that empower breakthroughs in gene therapy research and development.
Introduction to AAV Packaging
Adeno-associated viruses (AAVs) have emerged as a versatile and powerful tool in the realm of gene therapy. These small, non-enveloped viruses with a single-stranded DNA genome have garnered significant attention due to their remarkable properties, including their ability to transduce both dividing and non-dividing cells, their low immunogenicity, and their capacity for episomal expression without integrating into the host genome.
Fig.1 Schematic of the genome organization of wild-type AAV. (Lee E. J., et al., 2018)
The process of AAV packaging involves the encapsidation of a recombinant AAV (rAAV) vector, which typically contains a promoter, a gene of interest, and the necessary inverted terminal repeat (ITR) sequences required for viral replication and packaging. This intricate process requires the coordinated expression of various viral genes, including the rep and cap genes, which are responsible for viral replication and capsid formation, respectively.
Types of AAV Packaging Systems
To streamline the production of recombinant AAV vectors, researchers have developed several advanced AAV packaging systems. These systems can be broadly divided into the following approaches:
Table 1. Comparison of different rAAV packaging systems. (Merten O. W., 2016)
| Production system |
Biological system used |
Cell specific production rate (vg/c) |
Volumetric production (vg/L) |
Largest scale used |
rcAAV production |
Percentage of full particles (%) |
Encapsidated rep/cap, HSV sequences (%) |
| Plasmid transfection |
Selected suspension adapted HEK293 clone |
1-2x105 (AAV2) |
1014 |
20L (WAVE)** |
+* |
5-50 |
Rep: 0.3-1.5
Cap:0.4-1; 0.016-0.024 |
| Stable cell line |
HeLa (rep-cap/rAAV vector), infection with wt adenovirus 5 |
>5x104*** |
>5x1013 |
250L (STR) -> 2000L (STR) |
Below detection level |
>50 |
|
| Herpes simplex type 1 |
Suspension adapted BHK cells, infection with 2 viruses (rHSV-rep2capX/rHSV-rAAV vector), MOI=4/2 |
6.9x104 to1.13x105 |
2.4x1014 (AAV1) |
25L (WAVE) -> 100L (WAVE) |
Below detection level |
97.55±0.24% |
HSV: 0.007-0.012 |
| Baculovirus system |
Sf9, infection with 2 viruses (Bac-rep2/capX/Bac-rAAV-vector), MOI=0.05/0.05 - 1.6/1.6 |
104 to 105 |
9.4x1013 (AAV1) |
200L (STR, WAVE) |
Below detection level |
10-40%**** (AAV8) |
Cap: 0.016 |
| OneBac 2.0 system |
Stable Sf9 cell line (rep-cap), infection with 1 virus (BAC-rAAV vector), MOI=5 |
~105 (AAV5) |
1.4x11515 |
Small scale** |
Below detection level |
No information |
Cap: 0.02
Rep: <0.001 |
*Depending on the plasmid system used. **Large production scale possible. ***Selection criteria. ****Depending on the ITR construct
Our Services
Protheragen, a leading provider of advanced gene therapy solutions, offers a comprehensive suite of AAV packaging services to support researchers and developers in the field of gene therapy. Our state-of-the-art facilities and experienced team of scientists are dedicated to delivering high-quality, customized AAV vectors tailored to the specific needs of our clients.
Methods of Traditional AAV Packaging
Triple Transfection-Based AAV Packaging
Our triple transfection-based AAV packaging service leverages the power of HEK293 cells to produce recombinant AAV vectors. We provide a streamlined process that involves the co-transfection of three plasmids: one containing the rAAV transgene, one with the necessary rep and cap genes, and one with the helper functions required for viral replication and packaging. This approach ensures efficient and reliable AAV production, with a typical turnaround time of 6-8 weeks.
Baculovirus-Mediated AAV Packaging
For clients seeking higher yields of recombinant AAV vectors, our baculovirus-mediated packaging service is the ideal solution. By utilizing the Sf9 insect cell system, we are able to generate large quantities of high-quality rAAV particles. This method involves the generation of recombinant baculoviruses carrying the rAAV transgene and the necessary rep and cap genes, followed by their co-infection in Sf9 cells. The resulting rAAV particles are then purified and characterized to meet the specific requirements of our clients.
AAVLink™ Packaging Platform
We streamline the process by incorporating all essential genes for AAV production into cells, allowing for the efficient generation of viral vectors through induction. This innovative approach addresses the limitations of the existing three-plasmid system, such as challenges in amplification, elevated impurities, and increased costs. Furthermore, we offer a highly effective dual AAV system for gene expression that surpasses the AAV packaging limit, enhancing the versatility and productivity of gene delivery strategies.
At Protheragen, we understand that every project is unique, and that's why we offer customized AAV packaging services to cater to the specific needs of our clients. Whether you require a particular AAV serotype, a specific promoter, or have unique vector design considerations, our team of experts will work closely with you to ensure the successful development of your tailored AAV vector. If you are interested in our services, please feel free to contact us for more details and quotation information of related services.
References
- Kontogiannis, Theodoros, et al. "Characterization of AAV vectors: A review of analytical techniques and critical quality attributes." Molecular Therapy Methods & Clinical Development 32.3 (2024).
- Merten, Otto-Wilhelm. "AAV vector production: state of the art developments and remaining challenges." Immuno-oncology Insights (2016).
