AAV Packaging Plasmids
BioInnovatise Viral Vector Team
Updated December 9, 2024
Molecular Cloning & Mutagenesis Projects Completed
Viral Vector Packaging Solutions Delivered
Plasmid DNA Preparations Successfully Produced
Packaging Plasmids Used In AAV Packaging
Typically there are 3 packaging plasmids used during AAV packaging, as noted in a typical AAV packaging protocol, which are used to efficiency create AAV vectors.
- Transgene Plasmid – containing the gene of interest flanked by AAV inverted terminal repeats, ITRs
- Rep/Cap Plasmid – provides AAV replication and capsid proteins
- Helper Plasmid – provides essential adenovirus functions (also referred to as pHelper)
The use of a pHelper plasmid allows for the supply of E2A, E4, and VA RNA genes as well as supporting viral replication. The above diagram illustrates the AAV packaging process at BioInnovatise where our team uses HEK293T cells for AAV packaging.
Rep/Cap Packaging Plasmid's Role
The Rep/Cap plasmid most significantly affects AAV packaging capacity. Rep/Cap plasmids affect:
- The efficiency of viral particle production
- The ability to package DNA
- The maximum insert size that can be packaged
- Capsid protein expression levels
pHelper Packaging Plasmid's Role
The vector serotype does slightly affect plasmid considerations. While the fundamental plasmid structure remains similar, the Rep/Cap plasmid must encode the specific capsid proteins for the desired serotype (e.g., AAV2, AAV8, AAV9). Each serotype has unique capsid protein variants that determine tissue tropism and transduction efficiency. Learn more about adeno-associated virus serotypes.
Does Serotype Affect Which Packaging Plasmids Are Used?
Definitely! This is perhaps the largest way to optimize your AAV vector for high performance (efficient delivery of the genetic payload to target cells).
Our team has made this quick list to help highlight important packaging plasmid considerations when packaging AAV vectors:
- Rep/Cap Plasmid Impact:
- The most crucial plasmid affecting titer levels.
- Rep protein levels directly influence viral genome replication efficiency, packaging process completeness
and overall viral particle production. - Different Rep/Cap variants can dramatically change titer levels
- Some serotypes, like AAV8, naturally produce higher titers compared to others.
- Plasmid Ratio Optimization:
- Incorrect plasmid ratios can severely reduce titer
- Typical optimal ratios:
- 1:1:1 to 1:1:3-4 (transgene:Rep/Cap:helper)
- Slight variations can increase or decrease titer by 2-10 fold
- Excess Rep proteins can actually inhibit viral particle formation.
- Other considerations may include looking at the promoter strength in the packaging plasmids, plasmid sequence integrity, and potential mutations in Rep/CAp genes.
AAV Packaging Capacity Considerations
Researchers who use AAV as their viral vector understand the low AAV packaging capacity they must work with. However, if your transgene plasmid is too large to be efficiently packaged, there are work arounds:
- Split the transgene into two separate AAV vectors – dual vector approach.
- Use AAV9 or AAV-DJ, which have slightly larger packaging capacities.
- Employ genome-based engineering techniques like removing non-essential sequences.
- Consider using Cre-lox recombination systems for complex gene delivery.
Do AAV Packaging Plasmids Effect Titer Levels?
Definitely! This is perhaps the largest way to optimize your AAV vector for high performance (efficient delivery of the genetic payload to target cells).
Our team has made this quick list to help highlight important packaging plasmid considerations when packaging AAV vectors:
- Rep/Cap Plasmid Impact:
- The most crucial plasmid affecting titer levels.
- Rep protein levels directly influence viral genome replication efficiency, packaging process completeness
and overall viral particle production. - Different Rep/Cap variants can dramatically change titer levels
- Some serotypes, like AAV8, naturally produce higher titers compared to others.
- Plasmid Ratio Optimization:
- Incorrect plasmid ratios can severely reduce titer
- Typical optimal ratios:
- 1:1:1 to 1:1:3-4 (transgene:Rep/Cap:helper)
- Slight variations can increase or decrease titer by 2-10 fold
- Excess Rep proteins can actually inhibit viral particle formation.
- Other considerations may include looking at the promoter strength in the packaging plasmids, plasmid sequence integrity, and potential mutations in Rep/CAp genes.
Notes from the laboratory at BioInnovatise:
- Our team uses a HEK293T cell line a highly efficient transfection AAV packaging cell line.
- We use RT-qPCR (Real-time polymerase chain reaction) to measure titer levels.
- If you are unsure your transgene is too large to be packaged efficiency, contact our molecular cloning and viral vector team for alternate solutions.
Trusted By Top Researchers Across Disciplines and Therapeutic Areas
Subscribe for Product Releases, New Resources, and Company Updates
13 Taft Court Suite 220
Rockville, MD 20850
(301) 838-8675
Follow Us on LinkedIn
© 2025 BioInnovatise, All Rights Reserved