Advancements in gene therapy have newly emerged with the increased demand for efficient and reliable AAV packaging services. Protheragen offers sophisticated scientific prowess along with high-touch customer service that guarantees the availability of AAV packaging services to catalyze advancements in gene therapy R&D.
Introduction to AAV Packaging
Adeno-associated viruses (AAVs) are gaining traction in the field of gene therapy and their applications. These small, non-enveloped viruses containing a single-stranded DNA genome receive attention due to their pronounced features, which consist of transducing both dividing and non-dividing cells, low immunogenicity, as well as episomal expression without incorporation into the host genome.
Fig.1 Schematic of the genome organization of wild-type AAV. (Lee E. J., et al., 2018)
AAV packaging begins with the encapsidation of a recombinant AAV (rAAV) vector, which consists of a promoter, a gene of interest, and inverted terminal repeat (ITR) sequences needed for viral replication and packaging. This process is quite elaborate in nature as it needs sequential expression of several genes, such as rep and cap, to produce the virus’s backbone and its structural proteins.
Types of AAV Packaging Systems
In order to increase the efficiency of producing recombinant AAV vectors, various sophisticated AAV packaging systems have been developed. These systems may be categorized 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 stands out in the market as a leader in AAV packaging services with specialized gene therapy solutions. We offer cutting-edge facilities and sophisticated scientific human resources, which provide unique customized services to clients in relation to their specifications concerning AAV vectors.
Methods of Traditional AAV Packaging
Triple Transfection-Based AAV Packaging
With our AAV packaging service, we apply a triple transfection strategy that utilizes HEK293 cells for the production of recombinant AAV vectors. We offer a streamlined process which includes the co-transfection of three plasmids; one with the rAAV transgene, another one containing rep and cap genes, and a third one encoding for the helper functions needed for viral replication and packaging. This method guarantees dependable and efficient production of AAV, typically completed in 6-8 weeks.
Baculovirus-Mediated AAV Packaging
Our baculovirus-mediated packaging service is best for clients looking to obtain higher yields of recombinant AAV vectors. Using the Sf9 insect cell system, we can produce large amounts of rAAV particles while maintaining quality. This process entails generating recombinant baculoviruses containing the rAAV transgene and the required rep and cap genes, then co-infecting them into Sf9 cells. We purify and characterize the rAAV particles so that they satisfy all client specifications.
AAVLink™ Packaging Platform
For the purposes of AAV-based viral vectors production, we have put into cells all the necessary genes so that viral vectors can be easily and efficiently produced by induction. This novel system overcomes some shortcomings of the existing three-plasmid system, which suffers from poor amplification, high impurities, and high costs. In addition, we provide a very efficient dual AAV systems for gene expression, which go beyond the AAV packaging limit, thereby increasing the flexibility and efficiency of gene delivery options.
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).
