AAVtri Three-Plasmid AAV Production System
The three-plasmid AAV production system is the most widely used and well-established platform for generating recombinant adeno-associated virus (AAV) vectors. This system relies on the co-transfection of three separate plasmids into producer cells, typically HEK293-based cell lines: (1) a transfer plasmid containing the gene of interest flanked by AAV inverted terminal repeats (ITRs), (2) a packaging plasmid encoding the AAV rep and cap genes, and (3) a helper plasmid providing essential adenoviral helper functions. Together, these components enable efficient AAV genome replication, capsid assembly, and vector production, making the three-plasmid system a standard approach for academic research and early-stage gene therapy development.
AAVtri System
AAVnerGene’s AAVtri system production involves the transfection of host cells with three separate plasmids:
- pAAVtri vector plasmid – Contains the gene of interest (GOI) flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs), which are essential for AAV genome replication and packaging. This plasmid serves as the template for recombinant AAV vector production.
- pRCap helper plasmid – Encodes the AAV rep and cap genes, which produce the replication and capsid proteins, respectively. Rep proteins mediate AAV genome replication and packaging, while Cap proteins drive capsid assembly. This plasmid provides the core viral components required for AAV vector production.
- mini-pHelper-1.0: One of our key developments in the AAVtri system is the mini-pHelper-1.0, which is a smaller size plasmid (8.4kb) that provides Ad helper functions. This plasmid contains the necessary adenoviral genes, such as, E2a, E4orf6, and VA RNA, which are required to support AAV replication and packaging. Replacement pHelper in traditional triple-plasmid with the mini-pHelper plasmid in our AAVtri system has shown significant improvements in AAV titer, ranging from 100% to 250% across various serotypes.
Design of mini-pHelper-1.0
The most commonly used and best-studied Ad helper functions originate from the human adenovirus type 2 or 5 (Ad2 or Ad5). Researchers have continually made efforts to reduce the size of Ad helper, such as pBHG10, pXX5, pXX6, and pAdDeltaF6. The minimal set of Ad helper factors for efficient AAV replication and production, consists of five Ad molecules: E2A, E4orf6, and the VA RNA. The widely used pHelper plasmid is developed based Ad2, which has a 11.6 kb total genome with 9.3 kb of Ad genome.
AAVnerGene has developed mini-pHelper-1.0, a streamlined helper plasmid that retains only 6.1 kb of the Ad2 genome, resulting in a compact total plasmid size of 8.4 kb. This innovative design incorporates three key modifications [US20240132911A1]
- Deletion introns of E2A
- Deletion introns of E4orf6
- Codon optimization of E2A
AAV packaging efficiency is a crucial parameter to consider in AAV vector production systems. Replacement pHelper with the mini-pHelper-1.0 plasmid has shown significant improvements in AAV titer, ranging from 100% to 250% across various serotypes [Rongze Yang et al., AAVone: A Cost-Effective, Single-Plasmid Solution for Efficient AAV Production with Reduced DNA Impurities. https://www.sciencedirect.com/science/article/pii/S2162253125001179].
Plasmids for AAVtri system
- Plasmids are offered either individually or as a bundled AAV packaging kit (e.g., mini-pHelper-1.0 + pRCap-AAV9 + pAAVtri-CMV-EGFP), with a 10% discount available for bundle purchases.
- A Material Transfer Agreement (MTA) is required for academic use of mini-pHelper-1.0 and its derivates, as well as pRCap plasmids, and a license agreement is required for industrial use)
mini-pHelper-1.0 and pHelper plasmids
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mini-pHelper-1.0 derivates:
mini-pHelper-AAVs is a compact, optimized plasmid designed to produce truly-empty AAV particles through transfection alone, or recombinant AAV vectors when co-transfected with pAAVtri plasmids. mini-pHelper-Rep is specifically used for the production of AAV capsid libraries.
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pRCap plasmids
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Notes:
- All the plasmids are ampicillin resistance (Amp).
- A Material Transfer Agreement (MTA) is required for academic use of pRCap plasmids, and a license agreement is required for industrial use.
- AAVnerGene has hundreds of pRCap plasmids with different AAV Caps.
pAAVtri plasmids
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