Traditional triple plasmid AAV production 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 helper 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): This plasmid contains the necessary adenoviral genes, such as, E2a, E4orf6, and VA RNA, which are required to support AAV replication and packaging. The AAV helper plasmid provides the trans-acting factors necessary for efficient AAV vector 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 Triple Plasmid System. Replacement pHelper with the mini-pHelper plasmid has shown significant improvements in AAV titer, ranging from 100% to 250% across various serotypes.
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[1][2]. The minimal set of Ad helper factors for efficient AAV replication and production, consists of five Ad molecules: E2A, E4orf6, and the VA RNA[3]. 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 created the mini-pHelper, which only remains 6.1 kb of Ad2 genome and with a total plasmid size 8.4 kb. Replacement pHelper with the mini-pHelper plasmid has shown significant improvements in AAV titer, ranging from 100% to 250% across various serotypes. The mini-pHelper and pHelper systems have been shown to generate similar AAV vectors in terms of the ratio of empty to full particles, as well as the AAV potency, capsid and genome components. Those findings support the use of the mini-pHelper to replace the pHelper for AAV production and for creating our AAVone system.
[1]Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.
[2]pAdDeltaF6 was a gift from James M. Wilson (Addgene plasmid # 112867)
[3]The Interplay between Adeno-Associated Virus and Its Helper Viruses.
AAV packaging efficiency is a crucial parameter to consider in AAV vector production systems. Replacement pHelper with the mini-pHelper plasmid has shown significant improvements in AAV titer, ranging from 100% to 250% across various serotypes.
Note: Optimization the molecular ratios of three plasmid(pRC:mini-pHelper:pGOI) is 0.75:1:1 or 1:1:1.
Absolutely, the quality of AAV vectors is a critical parameter to consider in AAV vector production systems. The mini-pHelper and pHelper systems have been shown to generate similar AAV vectors in terms of the ratio of empty to full particles, as well as the AAV potency, capsid and genome components.
PacBio Sequencing depth across the AAV genome showed no difference among the mini-pHelper-triple plasmid system, phelper-triple plasmid system and AAVone system.
The empty-to-full ratio is an important parameter to consider in AAV vector production. It refers to the ratio of AAV particles that do not contain a therapeutic payload (empty capsids) to those that carry the desired gene of interest (full capsids). Ideally, a high-quality AAV vector preparation should have a low empty-to-full ratio, indicating a high proportion of full capsids. This is desirable because empty capsids do not contribute to the therapeutic effect and may occupy space that could otherwise be utilized by full capsids.
In the mini-pHelper based triple plasmid system, where the pHelper plasmid is replaced with the mini-pHelper plasmid, it has been observed that the empty-to-full ratio of AAV vectors is similar to that of the original pHelper system.