AAV Genomic Integrity
AAV Genomic Integrity
Genomic integrity refers to the completeness and accuracy of the AAV genome, which includes the transgene cassette and the flanking inverted terminal repeats (ITRs). The ITRs are critical for replication, packaging, and integration of the viral genome, while the transgene cassette contains the therapeutic gene and regulatory elements necessary for its expression. Any alterations, deletions, or rearrangements in the AAV genome can compromise the functionality of the vector, leading to reduced therapeutic efficacy or even safety concerns.
AAV genomic integrity is not just a technical detail—it is the foundation of successful gene therapy. By ensuring that AAV vectors deliver complete and accurate genomes, researchers and manufacturers can maximize therapeutic efficacy, minimize safety risks, and bring life-changing treatments to patients. As the field evolves, continued focus on improving genomic integrity will be essential to unlocking the full potential of AAV-based gene therapies.
To address this, AAVnerGene provides a variety of advanced analytical methods to assess AAV genomic integrity, including TapeStation, denaturing gel electrophoresis, charge detection mass spectrometry (CD-MS), PacBio or Nanopore next generation sequencing (NGS).
Price and Turnaround
| Method | Purpose | Turnaround | Price | Sample Requirement |
| Denature Gel | Size assessment | 1-2 day | $200/sample | ~100 μl of sample with titer >1E+12vg/ml |
| Tapestation | Size assessment | 1-2 day | $200/sample | ~100 μl of sample with titer >1E+12vg/ml |
| CD-MS | Size assessment | 3-4 weeks | Request | ~100 μl of sample with titer >1E+12vg/ml |
| PacBio Sequencing | Sequence details | 3-4 weeks | Request | ~100 μl of sample with titer >1E+12vg/ml |
| Nanopore Sequencing | Sequence details | 2-3 weeks | Starting from $600 | ~100 μl of sample with titer >1E+12vg/ml |
Choosing the Right Methods for AAV Integrity Analysis
- Denaturing Agarose Gel Electrophoresis
This traditional, cost-effective, and easy-to-use method allows visualization of AAV genomes under denaturing conditions. However, it is time-consuming and offers limited precision, making it less suitable for detailed analysis. - Southern Blotting
Southern blotting can confirm the presence and size of the target gene within the AAV genome. Despite its historical utility, this method suffers from low sensitivity and precision, and cannot detect genome fragments lacking the target sequence. - Agilent Tapestation
Tapestation is a semi-automated capillary electrophoresis system that offers improved speed and resolution over gel-based methods. It provides size distribution profiles of AAV genomes but may not detect low-abundance species or distinguish full vs. partially packaged genomes. - qPCR and ddPCR
Quantitative PCR (qPCR) and digital droplet PCR (ddPCR) are widely used for quantifying specific AAV genome sequences. While highly sensitive and accurate for titer determination, these methods do not provide information on genome size or detect truncated or rearranged genomes lacking the target sequence. - Charge Detection Mass Spectrometry (CD-MS)
CD-MS enables high-resolution, single-molecular analysis of AAV vectors by simultaneously measuring the mass and charge of individual genomes (after extraction), allowing for direct assessment of genome heterogeneity. In addition, CD-MS can distinguish full, empty, and partially packaged capsids through direct particle measurement of AAV vectors. However, its routine use is currently limited by the limited availability of commercial instruments.. - Next-Generation Sequencing (NGS)
NGS provides comprehensive sequence information, including the identification of genome integrity, mutations, and contaminants. While powerful, NGS remains too complex and costly for routine QC in most biomanufacturing workflows. PacBio Sequencing PacBio’s Single-Molecule Real-Time (SMRT) sequencing provides long-read capabilities ideal for analyzing full-length AAV genomes. It can accurately identify structural variants, truncations, and rearrangements. However, its relatively high cost may restrict widespread implementation in routine QC workflows.
Nanopore Sequencing Nanopore sequencing enables real-time, portable, and cost-effective long-read analysis of AAV genomes. It can reveal full-length genome structure and heterogeneity across vector populations. Despite its affordability and accessibility, the technology currently lacks the base-calling accuracy and consistency required for standard QC applications.
AAV Genome Heterogeneity
Early studies of wild-type AAV (wtAAV) revealed that not all assembled viral particles contained a complete genome. These were termed defective interfering (DI) AAV particles, characterized by reduced optical density and enrichment for sequences corresponding to the terminal regions of the AAV genome. When added to cultures replicating wtAAV, DI particles were shown to suppress the production of infectious virions and full-length AAV DNA.
Similar subgenomic particles have also been observed in recombinant AAV preparations:
Snapback Genomes (SBGs): Double-stranded DNA fragments containing either the 5′ or 3′ ITR. These can be symmetric (fully complementary) or asymmetric (lacking complementary sequence on one strand). In wtAAV, SBGs may play a regulatory role in replication, though their precise function remains unclear.
Incomplete Single-Stranded Genomes: Contain only one ITR (5′ or 3′) and lack the full-length genome.
Internally Deleted Genomes: Contain both ITRs but are missing internal sequences of the expression cassette, potentially resulting in truncated or aberrant gene products.
Mutated Genomes: A further source of genome impurity includes vector particles carrying mutated genomes. NGS technologies, such as PacBio Sequencing, have revealed sequence deviations from the intended construct.
The design of the expression cassette significantly impacts the prevalence of incomplete genomes. Constructs containing high secondary structure—such as short hairpin RNA (shRNA) or CRISPR single-guide RNA (sgRNA)—frequently lead to premature genome truncation during packaging. Termination hotspots have been identified in structured regions like the CMV enhancer, CB promoter, and EGFP gene, further implicating sequence architecture in genome resolution issues.
AAV genome analysis by TapeStation.
AAV genome truncation hotspots analysis by PacBio sequencing
AAV Genome Optimization
At AAVnerGene, we not only offer comprehensive AAV genomic integrity analysis, but also specialize in custom AAV genome design, optimization, and production based on these analytical insights. Our integrated approach supports both research and clinical development by ensuring each vector is tailored for maximum performance. Our expertise focuses on:
AAV Efficiency and Specificity – Enhancing targeted delivery and transduction across various tissues and cell types.
AAV Productivity and Quality – Achieving high-yield AAV production with rigorous quality control to ensure consistency and regulatory compliance.
Overcoming Size Limitations – Designing optimized constructs that maximize payload capacity while maintaining packaging integrity and functional expression.
Whether you’re developing a novel therapeutic or scaling up for IND-enabling studies, AAVnerGene provides the tools and support to accelerate your AAV program.