AAV Integrity Analysis by TapeStation
TapeStation
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.
Utilizing an automated electrophoresis system like the TapStation, combined with suitable denaturation conditions, allows for efficient and accurate evaluation of AAV ssDNA. This provides valuable insights into the purity and integrity of the AAV genome. This method will significantly contribute to the field by streamlining the AAV analysis process and improving the accessibility of quality control.
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 |
AAV Genomic Integrity Analysis by TapeStation
Analysis of synthesized ITR oligonucleotide. (A) Sequences of the synthesized ITR-oligo1–50, ITR-oligo1–100, and ITR-oligo1–150. (B) ITR-oligos were analyzed after addition of an equal amount of HS RNA sample buffer into oligo (10 ng/μL) with or without heat treatment at 75°C for 5 min. ITR-oligo1–50 without (lane 2) and with heat treatment (lane 3). ITR-oligo1–100 without (lane 4) and with (lane 5) heat. ITR-oligo1–150 without (lane 6) and with (lane 7) heat. An equal volume of HS RNA sample buffer was added to a 50-kb DNA ladder (10 ng/μL) with heat treatment at 75°C for 5 min (lane 1); 0.5 volume of HS RNA sample buffer was added to the HS RNA ladder, and the mixture was heated at 72°C for 3 min (lane 8). {Huaman Gene Ther. 2024 Feb 15;35(3-4):104–113. doi: 10.1089/hum.2023.148}
Analysis of AAV genomes extracted from purified AAV vectors produced from different production systems (pTri, tri-plasmid transfection of standard plasmids, mTri, tri-transfection of mini pHelper, and AAVone single-plasmid system. Automated electrophoresis analysis (TapeStation) of vector DNAs illustrating full-length genomes (arrow) and partial genomes (bracket). Arrow heads designate specific bands identified by software.