Integrated AAV Offerings: From Cutting-Edge Products to Advanced Custom Services

Traditional AAV Production Systems

One widely used method for producing AAV vectors involves co-transfecting HEK293 cells with three plasmids containing the necessary cis and trans components - pHelper, pRep-Cap, and pAAV. The two inverted terminal repeats (ITRs) serve as the cis elements required for AAV replication and packaging. Rep78 and Rep52 are non-structural proteins encoded in the AAV genome 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 also 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. 

AAV vector production 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. 

Our partner, AAVnerGene has 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 (AAVtri^TM) system with higher package efficiency compared to other Ad Helper plasmids. Based on the mini-pHelper, AAVnerGene has developed  a two-plasmid (AAVdual^TM) and a  single plasmid (AAVone^TM) system for AAV production.
 

Single-plasmid AAVone System

In the AAVone System, all the Ad helper genes (E2A, E4orf6 and VA RNA) (pHelper), AAV helper genes (Rep and Cap) (pRep-Cap), and AAV vector genome (pAAV) are assembled into one compact plasmid (pAAVone) with 13 kb oversized backbone. The selection marker is Kana. AAV vectors can be easily, efficiently and consistently generated by transfection of a single-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. It also increases full-to-empty ratio by ~10% in crude samples. AAVone system not only reduces the plasmid number from 3 to 1, but also reduces the total plasmid amounts from 1 to 0.25 ug per 1 million cells used for each production.

View our in-house pAAVone plasmids

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