AAV Packaging Limit
BioInnovatise Viral Vector Team
Updated December 9, 2024
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AAV packaging is particular challenging despite AAV being the prominent viral vector in cell and gene therapy, immunology, and vaccine development. With a low cargo limit, adeno-associated virus, requires researchers to use a smaller transgene / GOI than a transgene which might be sufficiently packaged in a adenoviral, lentiviral, or retroviral vector.
Recent research in AAV vector design and improvement have made some strides to maximizing the current AAV packaging limit and expanding it. However there are limits to how much plasmid can be sufficiently packaged while delivering the genetic payload to the target cells.
In this article, we will explore the current AAV packaging limit of 4.7 kb¹ while explaining how this finite space can be best used to deliver a genetic payload effectively.
If you are interested in learning about lentivirus packaging size limit, retrovirus packaging size limit or adenovirus packaging capacity, we have created articles on those viral vectors as well.
Download our Viral Vector (AAV, Adenovirus, Lentivirus, and Retrovirus) Packaging Size Limit Guide
The Current AAV Packaging Limit
AAV vector genomes are been limited to 4.7 kb in length in order to balance the need for larger genetic constructs and effective payload delivery. Larger constructs can be attempted to be packaged in AAV vectors, however they are unlikely to be transfected and packaged enough to deliver the intended result. Constructs that are larger than 5 kb have no guarantee of a high titer (≥10E11 GC/ml). If you do have a larger construct and would like it shortened with molecular cloning, contact our team.
Some researchers have opted to trying a dual vector approach for larger AAV constructs.
Dual Vector Approach
The dual vector approach is one of the workarounds researchers have employed to get around AAV packaging limit challenges and lower integration stability.
Here is a brief overview on the two leading AAV dual vector approach strategies:
- Trans-Splicing Approach
- Vector 1 carries part of the transgene.
- Vector 2 carries the complementary part.
- Vectors co-infect the same cell.
- Cellular machinery splices the two partial mRNAs.
- Produces a full-length functional protein.
- Challenges:
- Low efficiency of splicing.
- Reduced overall expression levels.
- Requires precise design of splice sites.
- Cre-lox Recombination Approach
- One vector carries a “split” transgene with loxP sites.
- Second vector carries Cre recombinase.
- Cre enzyme facilitates complete gene reconstruction.
- Advantages:
- Higher recombination efficiency.
- More predictable gene reconstruction.
- Works well for complex genetic systems.
AAV Packaging Limit By Serotype
Tissue tropism is one of the biggest reasons researchers choose to use AAV as a viral vector. Each serotype does have has distinct cellular entry preference, immunological characteristic, and capsid protein composition. These different serotypes do have slightly differing AAV packaging limits for the GOI:
| AAV Serotype | Packaging Limit |
|---|---|
| AAV1 | 4.7 kb |
| AAV2 | 4.7 kb |
| AAV5 | 4.7 kb |
| AAV6 | 4.7 kb |
| AAV8 | 4.8 – 5.0 kb |
| AAV9 | 5.0 – 5.2 kb |
| AAVDJ | 5.3 – 5.5 kb |
| AAVrh10 | 4.8 – 5.0 kb |
| AAnc80 | 5.2 – 5.5 kb |
What Factors Affect The AAV Packaging Limit?
There are several main factors that can affect the packaging size limit of an AAV vector. However compared to more complex and evolved vectors such as lentivirus, the largest constraint for AAV packaging continues to be the overall small vector size.
- Natural packaging limit: AAVs have an inherent packaging capacity of approximately 4.7-5.0 kb, determined by the size of the wild-type AAV genome.
- ITR sequences: The inverted terminal repeats necessary for packaging occupy ~300 bp, reducing the available space for your transgene.
- AAV serotype: While most serotypes have similar packaging limits (see above chart), minor variations exist between serotypes.
- DNA secondary structure: Complex secondary structures or high GC content may reduce packaging efficiency.
- Regulatory elements: Promoters, enhancers, and polyA signals consume valuable space within your construct.
What Should I Do If My Plasmid DNA Construct Is Too Long To Package In AAV?
Our molecular cloning and viral vector teams work closely together on projects where transgene plasmids need to be modified to be packaged efficiently. However there are a number of solutions if a plasmid construct is too large to be packaged:
- Dual AAV systems: See above
- Minigene approach: Create a shortened version of your gene retaining essential functional domains.
- Swap current regulatory elements for compact regulatory elements: Use smaller promoters (e.g., minimal CMV), compact polyA signals, and efficient enhancers.
- Codon optimization: Optimize codons for expression while potentially reducing sequence length.
- Remove non-essential elements: Identify and remove non-critical sequences from your construct via gene synthesis or molecular cloning.
Do Packaging Plasmids Affect The AAV Packaging Limits
Packaging plasmids (containing Rep and Cap genes or pHelper) don’t directly affect the packaging limit of a transgene. However specific Rep and Cap genes used determine the AAV serotype produced, which can have minor effects on packaging efficiency. The fundamental constraint remains the physical capacity of the AAV capsid, which is approximately 4.7-5.0 kb regardless of the packaging system used.
If you have additional questions or concerns whether or not your AAV plasmid will be effectively packaged into a vector, contact our team.
Learn about our quick turnaround AAV packaging service.
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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