AAV Genome Packaging
BioInnovatise Viral Vector Team
Updated December 16, 2024
As a viral vector, adeno-associated virus, delivers a single-stranded DCA payload to the target cell unlike retrovirus and lentivirus which are RNA payloads (we overviewed the differences between AAV vs lentivirus in another article).
The AAV genome is a critical part of understanding this vital vector because of AAV’s low packaging capacity. There are several typical challenges for AAV genome packaging and most importantly, several strategies to overcome these challenges during the AAV packaging process.
Key Challenges in AAV Genome Packaging:
AAV vectors, depending on the serotype used, generally have a cargo capacity of 4.7 kilobases. In the AAV packaging process, there are often 3 plasmids used during transfection and only 1 is the transgene / GOI, making the limit cargo space of 4.7 kb even smaller for a transgene.
The genome structure must contain two ITRs (Inverted Terminal Repeats) at the end as well as 2 ORFs to express replication and capsid design (Rep / Cap). These two mandatory factors further reduce the available transgene size as well. If you are interested in different ORFs or Rep/Cap sequences for your AAV plasmid, our molecular cloning team can help.
Size Limitations: Theoretically, the AAV packaging capacity can range from ~4.8 – 5.0 kb, however in order to efficiently reach a high titer, getting close to the upper range limit can be difficult. There is a significant drop-off in AAV packaging efficiency close to and beyond 5.0 kb.
Structural Constraints: AAV packaging requires ITRs and rep/cap pHelper plasmids which limits the remaining cargo space for the transgene / GOI.
Packaging Mechanism Challenges: During the AAV packaging process, the AAV genome must fold into a very specific configuration to maintain structural integrity, fit within the small capsid, and eventually ensure efficient viral assemble and infection of the target cell. If the genome is unable to be packaged and delivered to the target cell efficiently, the effect on the target cell and research model or patient is lost.
Effect Of Genome Size On AAV Vector Packaging
Because of the previously mentioned limited cargo capacity of the AAV vector, the genome size of the AAV plasmid has a direct relationship to the inefficiency of viral packaging (i.e. the larger the AAV genome, the less likely the virus will titer well). We have constructed this table (for education purposes) that outlines the relationship between AAV genome size and packaging efficiency:
| AAV Genome Size: | Packaging Efficiency: | Difficulty Level: |
|---|---|---|
| < 4.0 kb | 100% | Very Easy |
| 4.0 kb | 95% | Easy |
| 4.5 kb | 80% | Moderate |
| 4.7 kb | 70% | Challenging |
| 5.0 kb | 50% | Difficult |
| 5.5 kb | 25% | Very Difficult |
| 6.0 kb | 10% | Nearly Impossible |
Strategies To Maximize AAV Genome Packaging
To maximize the genome size for efficient AAV packaging, we recommend a few strategies:
- Genome Design Optimization: Minimizing unnecessary sequences in the transgene, using smaller promoters, and eliminating regulatory elements can increase viral titering.
- Dual Vector Approach: Dividing larger transgenes, such as CAR-T expressing transgenes, across AAV vectors to deliver the entire desired genetic payload.
- Advanced Packaging Techniques: Certain helper-free packaging systems, AAV packaging cell lines, and next-gen serotypes can accommodate larger genome sizes.
If you’re unsure your AAV plasmid will be efficiently packaged into an AAV vector, contact our team for a consultation.
Learn about our quick turnaround AAV packaging services.
Want to learn more about the latest in AAV based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News are always collecting and publishing the latest information on AAV based research.
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