1. ITR Structure and Function
• ITRs are crucial: ITRs are key elements in AAV vectors, serving as origins of replication, and are essential for viral genome replication, packaging into capsids, and vector persistence.
• Palindromic regions: AAV ITRs have a characteristic T-shaped structure composed of palindromic regions (A-A’, B-B’, and C-C’).
• C-C’ region deletion: The mentioned 11-nt deletion (GCCCGGGCAAA) occurs within the C-C’ palindromic region of the ITR.
2. Implications of the GCCCGGGCAAA Deletion
• Reconstitution during replication: Importantly, this specific 11-nt deletion is reported to be reconstituted during AAV replication, meaning the complete ITR sequence is restored in the viral genome.
• Minor impact on AAV production: Studies suggest that this deletion, despite its presence in the plasmid, does not significantly affect AAV production.
• Self-repair mechanism: ITRs have a unique ability to self-repair during AAV replication, potentially through the intact ITR on the opposite end of the genome. This could explain the reconstitution of the full sequence despite the initial deletion.
3. ITR Instability and Mutations
• High GC content: ITRs are known for their instability due to their palindromic nature and high GC content, making them prone to deletions during bacterial propagation.
• Common deletions: Small deletions in the B or C loop regions of ITRs are frequently observed during plasmid amplification.
• Challenges in AAV work: ITR instability poses challenges in AAV vector production and can affect vector yield, packaging efficiency, and even gene expression.
In Summary: The GCCCGGGCAAA deletion is a specific example of an ITR deletion commonly observed in AAV plasmids. Although it occurs in the C-C’ region, it is often reconstituted during AAV replication, and generally has a minimal impact on AAV production but increases transgene expression. This underscores the dynamic nature of ITRs and their unique self-repair mechanisms.