AAV Packaging Signal
BioInnovatise Viral Vector Team
Updated March 3, 2025
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The AAV packaging signal plays an important role when using adeno-associated vectors for gene delivery. Intact and optimized AAV packaging signals have impact on vector design, vector capacity, manufacturing/packaging efficiency, vector purity, and stability.
Our viral vector team has created this resource on the AAV packaging signal to summarize what AAV packaging signals are, their role in vector assembly, hazards to avoid in packaging signals. Let’s get into it!
AAV Vector Structure
What Are Packaging Signals? Where Are They In the AAV Genome?
In viral vectors such as AAV, adenovirus, lentivirus, and retrovirus, viral packaging signals are specialized genetic elements that serve as molecular “address tags” to ensure viral genomes are correctly packaged into newly formed viral capsids. These signals are critical for the virus life cycle.
For AAV vectors, the AAV genome contains a packaging signal located within the inverted terminal repeats (ITRs) at both ends of the AAV genome. ITRs are 145 nucleotides long with palindromic sequences that form T-shaped hairpin structures. These structures serve as origins of replication and packaging signals. The specific sequence within the ITR, is called the packaging recognition sequence (PRS), which is recognized by Rep proteins. Many researchers who use AAV vectors are arledy familiar with Rep/Cap plasmids because of their role during AAV packaging for capsid assembly.
AAV Packaging Signal Position: The packaging signals in AAV are located within the ITRs at both ends of the viral genome.
Size and Structure: AAV packaging signals are ~145 bp long, form hairpin T-shaped structures, and include:
- Rep Binding Element (RBE): 5′-GAGCGAGCGAGCGCGC-3′
- Terminal Resolution Site (trs): 5′-GTTGG-3′
- Secondary Rep Binding Element (RBE’)
Genome Context: The AAV genome (~4.7 kb) is much smaller than the adenoviral genome, between the ITRs are the viral genes responsible for capsid assembly (Rep/Cap), and in recombinant AAV, these genes are replaced with therapeutic payloads.
The above diagram overviews the differences between AAV packaging signals and adenovirus packaging signals within their respective vector genomes. Both genomes are flanked by ITRs however the AAV genome has T-shaped harpin structure and a smaller packaging signal at both ends.
The Role Of AAV Packaging Signals During AAV Packaging and AAV Manufacturing
During the vector assembly phase of AAV packaging, the Rep proteins (particularly Rep78 and Rep68) recognize the packaging sequences within the ITRs. These Rep proteins form a packaging complex that interacts with the capsid proteins. The complex facilitates DNA translocation into the preformed capsid through Rep’s ATP-dependent helicase activity.
AAV packaging signals play their role during the above “Packaging Using HEK293T cell line” phase of the AAV packaging process.
Cell And Gene Therapy Applications
AAV vectors continue to be useful in cell and gene therapy applications as well as other areas of biopharmaceutical medicine. Researchers who use adeno-associated viral vectors to deliver genetic payloads to target cells should understand the unique nature of AAV packaging signals vs. adenovirus packaging signals, or other vector packaging signals in their research applications including:
- ITRs are the only cis-acting elements required for packaging. This allows most viral genes to be removed, creating space for therapeutic genes
- Different AAV serotypes have slightly different packaging requirements
Can Something Go Wrong With Adenovirus Packaging Signals?
Unfortunately yes, packaging signals can definitely go bad and cause problems. Several types of issues can occur with viral packaging signals:
- Mutations in the ITRs can reduce or eliminate packaging efficiency.
- Deletions in the Rep binding elements (RBE) or terminal resolution site can prevent proper DNA packaging.
- Structural changes that disrupt the T-shaped hairpin formation can severely impair packaging.
- Even single nucleotide changes in critical regions can reduce packaging efficiency by 10-100 fold.
If there are problems in the packaging signal, the AAV packaging process is likely to have a reduced total yield of viral particles, incomplete/fragmented genomes are packaged, off-target packaging may include to packaging of non-viral DNA and therefore contamination with host cell DNA or helper virus sequences, and specific to AAV, ITR defects can lead to packaging of genome concatemers thus exceeding the capacity of the capsid and causes structural instability.
How To Mitigate AAV Packaging Signal Errors
There are a few ways researchers and viral vector manufactures can reduce AAV packaging signal errors including:
- Using specialized bacterial strains for propagating ITR-containing plasmids.
- Implementing quality control assays to verify packaging signal integrity.
- Engineering packaging signals with redundant elements to maintain function despite minor mutations.
If you have questions or concerns about packaging your AAV GOI, contact our viral vector 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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