AAV Production Systems

AAV vectors have become a preferred choice for gene delivery due to their efficient transduction, long-term gene expression, low immunogenicity, and favorable safety profile. They have been successfully utilized in numerous clinical trials and have received regulatory approvals as drugs for human use. While various systems have been proposed for AAV vector production, scaling up production to meet the demands of basic research in large animal models and clinical applications poses several challenges. These challenges include ensuring high production yields, maintaining vector purity, optimizing upstream and downstream processes, and complying with regulatory guidelines and Good Manufacturing Practices (GMP).
One widely used method for producing AAV vectors involves co-transfecting HEK293 cells with three plasmids containing the necessary cis and trans components. The two inverted terminal repeats (ITRs) serve as the only cis elements required for AAV replication and packaging. Among the four non-structural proteins encoded in the AAV genome (Rep78, Rep68, Rep52, and Rep40), only Rep78 and Rep52 are necessary as trans elements for AAV production. The capsid proteins VP1, VP2, and VP3 play a crucial role in AAV capsid assembly and are therefore required as trans elements. To facilitate AAV production, helper viruses like adenovirus (Ad) and herpes simplex virus (HSV) are used to provide the extra trans-acting factors required. 
The AAV vectors produced using the traditional transient transfection method have demonstrated safety, convenience, and effectiveness in clinical trials. However, this method has limited scalability potential. It is necessary to transfect all three plasmids into a cell, which limit the efficiency of AAV vector production. Transfecting multiple plasmids simultaneously can result in competition for cellular resources and introduce variability between production batches due to fluctuations in plasmid quality. Thus, optimizing the ratio of the three plasmids is crucial step for achieving higher AAV yields, especially for GMP grade AAV production. Moreover, producing of three GMP grade plasmids with greater than 95 percent purity and free from process-related impurities and variants, remains a challenge and is  time and labor cost. 
AAVnerGene developed innovative technologies to improve 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 Triple Plasmid System, with higher package efficiency compared to other Ad Helper plasmids. Based on the mini-pHelper, AAVnerGene has developed  a Dual Plasmid System  and an  single plasmid system, AAVone System  for AAV production. 
AAVone production systems

Production Systems


Plasmid Size(kb)

Total Genome Size(kb)

 Plasmid Number

Package Efficiency

AAVone System






Dual Plasmid System








Triple Plasmid System










Tranditonal Triple Plasmid System










AAVone System

AAVone production 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 used for each production.

Dual plasmid system

In the Dual Plasmid System, the AAV genome with transgene is assembled into our novel mini-pHelper plasmid to form the pAAVdual 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.
The Dual Plasmid System:
           ---- 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. 

In AAVnerGene's Triple Plasmid 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.