AAV Packaging Capacity

BioInnovatise Viral Vector Team

Updated December 9, 2024

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 capacity, 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 capacity 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 capacity 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.

The Current AAV Packaging Capacity

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 capacity 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.