AAVdual System

Traditional three-plasmid system used for AAV vector production present challenges when scaling up the process. The AAVdual System are developed based on our novel mini-pHelper plasmid.  In this system, the AAV genome with transgene is cloned into mini-pHelper and get pAAVdual-GOIs. AAV vectors can be generated by co-transfection pAAVdual-GOIs with the regular AAV helper plasmid. This system offers high  flexibility and high AAV packaging efficiency in AAV production. 

Traditional Triple Plasmid System involves the transfection of host cells with three separate plasmids:

  • AAV Vector plasmid(pGOI): This plasmid contains the gene of interest (GOI) flanked by inverted terminal repeats (ITRs), which are essential for AAV replication and packaging. The AAV vector plasmid serves as the template for the production of AAV vectors.
  •  AAV Rep-Cap plasmid(pRep-Cap): This plasmid carries the Rep and Cap genes, which encode the replication and capsid proteins of AAV, respectively. The Rep proteins are responsible for AAV genome replication and packaging, while the Cap proteins are involved in AAV capsid assembly. The AAV Rep-Cap plasmid provides the necessary machinery for AAV vector production.
  • Ad helper plasmid(pHelper): The Ad helper plasmid contains the necessary adenoviral genes, such as, E2a, E4orf6, and VA RNA, which are required to support AAV replication and packaging. The Ad helper plasmid provides the trans-acting factors necessary for efficient AAV vector production
By employing different combinations, it is possible to create three different types of dual plasmid systems for AAV production[1,2,3]:
  • Dual-V1: Combine Ad helper with AAV helper, such as pDG and pQT system
  • Dual-V2: Combine Ad helper with pGOI, such as AAVnerGene’s pAAVdual system
  • Dual-V3: Combine pGOI with AAV helper, such as pOXB system
The cost of raw materials plays a significant role in the economics of AAV manufacturing, especially in large-scale production for clinical and commercial purposes. Using a dual plasmid system instead of a three-plasmid system can offer clear advantages in terms of reducing the cost of goods for AAV production. 

  • Simplified Production: A Dual plasmid system streamlines the manufacturing process by requiring fewer plasmids to be produced, purified, and quality-controlled. This simplification can lead to cost savings in terms of materials, labor, and time.
  • Reduced Complexity: With fewer plasmids to handle, there is a reduced risk of errors in the manufacturing process, which can be costly to correct. Fewer components also make the production process more manageable.
  • Lower Raw Material Costs: Plasmids are among the most expensive raw materials in AAV production. By reducing the number of plasmids needed from three to two, the overall raw material costs can be significantly reduced, contributing to a more cost-effective manufacturing process.
  • Scalability: Cost savings achieved through a two-plasmid system can become even more pronounced as AAV production is scaled up for larger batches. The reduced complexity and lower material costs can make large-scale manufacturing more economically viable.

While all dual-plasmid designs offer above benefits, only Dual-V2, as exemplified by AAVnerGene’s pAAVdual system, seems to offer a balance between package efficiency and flexibility. 

  • Dual-V1 maintains flexibility in producing different AAV serotypes, which can be valuable for researchers working on a range of applications. However, it may exhibit lower package efficiency, potentially due to the complexity of combining Ad and AAV helper plasmids.
  • Dual-V3: Dual-V3 may offer increased AAV package efficiency, which is essential for maximizing the yield of properly packaged AAV vectors. However, it comes at the cost of reduced flexibility because it couples the GOI with the AAV helper plasmid, limiting the ease of switching and customizing the GOI.
  • Dual-V2, represented by AAVnerGene’s pAAVdual system, stands out by maintaining both package efficiency and flexibility. It allows researchers to work with various AAV serotypes while ensuring that the AAV vectors are efficiently packaged. This balance can be particularly advantageous when working on diverse gene therapy or vector biology projects.

AAVdual System is a dual plasmid AAV production system, in which the mini-pHelper was combined with the gene of interest (GOI).

Different with other dual plasmid systems, AAVnerGene’d AAVdual is build based on our novel mini-pHelper plasmid. Replacement pHelper with the mini-pHelper plasmid has shown significant improvements in AAV titer, ranging from 100% to 250% across various serotypes in triple plasmid systems. By incorporating the mini-pHelper and the GOI into a single plasmid, the AAVdual system simplifies the AAV production process and offers several advantages. 
  • Reduce Plasmid Number.  It reduces the number of plasmids required for transfection from 3 to 2. In traditional AAV production systems, three separate plasmids are needed to deliver the Rep and Cap genes, AAV helper genes, and the GOI. By combining the mini-pHelper and GOI into one plasmid, the AAVdual system eliminates the need for an additional plasmid, simplifying the transfection process.
  • Streamline the workflow. With fewer plasmids to handle and co-transfect, the process becomes more straightforward and less time-consuming. This can save both labor and resources during AAV production.
  • High package efficiency. The AAVdual system has been designed to exhibit high efficiency in generating AAV vectors. The integration of the essential elements for AAV replication and packaging in the mini-pHelper, along with the GOI, ensures robust and reliable AAV vector production.
  • Keep the flexibility.  The AAVdual system provides flexibility in producing different AAV serotypes. By choosing the appropriate Cap genes of the desired AAV serotype, researchers can easily generate AAV vectors with different serotype specificities using the same AAVdual system.
  • Compact plasmid size. The pAAVdual and pAAVdual-sc plasmids used in AAVdual system has size 11±2kb, making it easier to handle and produce during the manufacturing process and would not significantly reduce the plasmid yield. The compact size of the pAAVdual plasmid also offers practical benefits and simplifies cloning procedures. 
pAAVdual-CAG-Transgene Series$999.00$1,899.00
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pAAVdual-CAMKII-Transgene Series$999.00$1,899.00
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pAAVdual-CMV-Transgene Series$999.00$1,899.00
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pAAVdual-EF1a-Transgene Series$999.00$1,899.00
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pAAVdual-GFAP-Transgene Series$999.00$1,899.00
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pAAVdual-hSyn-Transgene Series$999.00$1,899.00
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pAAVdual-NSE-Transgene Series$999.00$1,899.00
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pAAVdual-sc-CMV-Transgene Series$999.00$1,899.00
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pAAVdual-sc-hSyn-Transgene Series$999.00$1,899.00
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  • We recommend to  use  pAAVdual:pRep-Cap at 2:1 molecular ratio for transfection.
  • AAVnerGene provides different templates(pAAVdual and pAAVdual-sc) for customers to clone their transgenes into our backbone.
  • AAVnerGene also provides DNA synthesis and clone services. Please contact us if you need a custom clone service.
  • All the plasmids are kanamycin  resistance(KanR)
  • A material transfer agreement(MTA) is required to  use any of mini-pHelper based plasmids, including pAAVdual plasmids.