The AAV packaging process involves the assembly of four main components, including Ad helper genes (pHelper, expressing E2A, E4orf6, and VA RNA), AAV helper genes (pRep-Cap, providing AAV Rep and Cap functions), AAV vector genome(pGOI), and a viral packaging cell line (such as HEK 293 and HEK 293T). Currently, co-transfection of the three plasmids into HEK293 cells is the most commonly used method for GMP-grade AAV production.
AAVnerGene developed innovative technologies for AAV production. One of our key developments is the mini-pHelper, which is a smaller size plasmid (8.4kb) that provides Ad helper functions in AAVtri System, with higher package efficiency compared to other Ad Helper plasmids. Based on the mini-pHelper, AAVnerGene has developed an AAVone™ single plasmid AAV production system and an AAVdual™ dual-plasmid system for AAV production.
| Production System | Plasmid Backbone | Ad Genes | Rep/Cap | Genome |
| AAVone System | pAAVone | Yes | Yes | ssAAV |
| pAAVone-sc | Yes | Yes | scAAV | |
| AAVdual System | pAAVdual | Yes | No | ssAAV |
| pAAVdual-sc | Yes | No | scAAV | |
| AAVtri System | pAAVtri | No | No | ssAAV |
| pAAVtri-sc | No | No | scAAV |
AAVone System
In the AAVone System, all the Ad helper genes (E2A, E4orf6 and VA RNA), AAV helper genes (Rep and Cap), and AAV vector genome are assembled into one plasmid (pAAVone). AAV vectors can be simply, efficiently and consistently generated by transfection of one plasmid into host cells. AAVone has demonstrated impressive results, achieving unpurified yields of over 1×10^15 viral genomes (VGs) per liter in suspension-cultured HEK-293T cells for most AAV serotypes, which is 2~4 fold higher than original triple plasmid transfection system. AAVone System not only reduce the plasmid number from 3 to 1, but also reduce the total plasmid amounts from 1 ug/million cells to 0.25 ug/million cells.
AAVdual System
In the AAVdual System, the AAV genome with transgene is assembled into our novel mini-pHelper plasmid to form the pAAVdual or pAAVdual-sc plasmid. AAV vectors can be flexibly and efficiently generated by co-transfection of pAAVdual plasmids with the regular AAV helper plasmids (pRep-Cap) into host cells.
---- Allows for modular design and flexibility in constructing AAV vectors.
---- Exhibits high efficiency in generating AAV vectors.
---- Streamlines the transfection process by reducing the number of plasmids.
AAVtri System
In AAVnerGene's AAVtri System, a small size version of Ad helper, mini-pHelper(8.4kb) was created and used to replace original pHelper plasmid. As the same as traditional triple plasmid system, AAV vectors are produced by co-transfection of mini-pHelper with pRep-Cap and pGOI plasmids into host cells. The reduction in size allows for more efficient transfection and reduces the overall cost of the process. Additionally, the increased packaging efficiency leads to higher yields of AAV vectors, which can be beneficial for applications such as gene therapy and gene editing where high-quality and high-titering AAV vectors are necessary.
pGOI: This plasmid contains the AAV inverted terminal repeats (ITRs) that flank the gene of interest (transgene) and all the necessary elements for packaging the transgene into AAV particles. The gene of interest can be inserted between the ITRs, and the whole construct is flanked by packaging signals that are essential for encapsidation during virus assembly.
pRep-Cap: This plasmid encodes the AAV replication and capsid proteins. The Rep proteins are involved in viral replication and regulation, while the Cap proteins form the outer capsid shell of the AAV particle. These proteins are required for packaging the AAV genome and producing infectious viral particles.
pHelper: This plasmid provides the necessary functions for AAV production but is not directly involved in the structure of the AAV particles. It typically contains the genes encoding the necessary adenoviral helper functions (E2A, E4orf6, and VA RNA).