In adeno-associated viruses (AAVs), the Inverted Terminal Repeat (ITR) sequences play a crucial role in the replication, packaging, and integration of the viral genome. ITRs are short, palindromic DNA sequences located at both ends of the single-stranded DNA genome.
AAV ITRs and their functions
Structure and Location: Wild-type AAV ITRs are typically around 145 base pairs in length, and they share a common stem-loop structure. They are located at both ends of the AAV genome, flanking the coding region. The ITRs are important for the stability and structural integrity of the viral genome.
Replication and Packaging: AAVs are replication-deficient on their own and require helper viruses (such as adenoviruses) to replicate their genomes. The ITRs serve as origins of replication for AAV when a helper virus is present. During the viral life cycle, the ITRs are recognized by viral and cellular factors, initiating DNA replication and transcription.
Packaging Signal: AAV ITRs contain a packaging signal that is necessary for the encapsidation of the viral genome into the AAV capsid. This packaging signal is recognized by viral proteins and is essential for the selective packaging of the AAV genome.
Integration and Latency: In the absence of a helper virus, AAVs can integrate their genomes into the host cell’s chromosomal DNA. The ITRs play a role in this site-specific integration process. The integrated AAV genome can persist in a latent state within the host cell, potentially leading to long-term expression of the integrated gene.
Stable Transgene Expression: AAV vectors used in gene therapy applications often replace the viral coding region with therapeutic genes of interest between the ITRs. These recombinant AAV vectors retain the ITRs, which provide the necessary elements for replication, packaging, and potential integration into the host genome.
Regulatory Elements: AAV ITRs contain various cis-acting elements that are important for gene expression, DNA replication, and encapsidation. These elements interact with cellular and viral factors to regulate these processes.
Engineering and Modification: Researchers have manipulated AAV ITRs to develop modified vectors with altered properties, such as improved packaging efficiency, tissue specificity, and reduced immunogenicity.
Serotype-Specific ITRs: Different AAV serotypes can have variations in their ITR sequences. These differences can influence the interactions of AAV with host factors, receptor binding, and tropism, contributing to the diversity of AAV serotypes and their applications.
Secondary structure of the AAV ITR
The AAV2 ITR serves as origin of replication and is composed of two arm palindromes (B-B' and C-C') embedded in a larger stem palindrome (A-A'). The ITR can acquire two configurations (flip and flop). The flip (depicted) and flop configurations have the B-B' and the C-C' palindrome closest to the 3' end, respectively. The D sequence is present only once at each end of the genome thus remaining single-stranded. The boxed motif corresponds to the Rep-binding element (RBE)  where the AAV Rep78 and Rep68 proteins bind. The RBE consists of a tetranucleotide repeat with the consensus sequence 5'-GNGC-3'. The ATP-dependent DNA helicase activities of Rep78 and Rep68 remodel the A-A' region generating a stem-loop that locates at the summit the terminal resolution site (trs) in a single-stranded form. In this configuration, the strand- and site-specific endonuclease catalytic domain of Rep78 and Rep68 introduces a nick at the trs. The shaded nucleotides at the apex of the T-shaped structure correspond to an additional RBE (RBE')  that stabilizes the association between the two largest Rep proteins and the ITR.
Flip and Flop
The ITR can occur in two alternative configurations—FLIP and FLOP. The two configurations arise from AAV replication, which can be predicted by a Cavalier–Smith model. The FLIP orientation has BB′ region closer to the 3′ end, and the FLOP has CC′ region at the same position. Two ITRs might flank the viral genome in all possible combinations: in mirrored FLIP/FLOP, FLOP/FLIP, and unidirectional FLIP/FLIP or FLOP/FLOP.
Understanding the ITR flip and flop is essential for unraveling the molecular mechanisms underlying AAV replication and for engineering AAV vectors with optimized properties for various applications, including gene therapy and basic research.
Goals of ITR Engineering:
- Improved Vector Performance: ITR engineering can lead to AAV vectors with enhanced properties, such as higher packaging efficiency, more stable transgene expression, and improved targeting of specific cell types or tissues.
- Enhanced Transgene Expression: ITR modifications can result in increased transgene expression levels, more sustained expression over time, or better control of gene expression in response to specific cues.
- Reduced Immunogenicity: Some modifications to the ITRs can help reduce the immune response triggered by AAV vectors, which is particularly important in gene therapy to minimize adverse reactions.
- Optimized Packaging Capacity: ITR engineering can potentially alter the packaging capacity of AAV vectors, allowing the incorporation of larger genetic payloads.
Strategies for ITR Engineering:
- Chimeric ITRs: Chimeric ITRs are generated by combining sequences from different AAV serotypes[Ling et al., 2016]. This can result in vectors with novel properties, as different serotypes have different receptor affinities and cellular entry mechanisms.
- Directed Evolution: Directed evolution involves creating libraries of AAV variants with randomly mutated ITRs. Variants with desired properties are then selected through iterative rounds of screening or selection.
- Rational Design: Rational design involves making targeted modifications to specific regions of the ITRs based on structural and functional insights, such as CpG modification[Pan et al., 2022]. This approach requires a thorough understanding of the ITR’s role in the viral life cycle. The deletion of the BB′ and CC′ regions also has a significant impact on AAV production. Modified U-shaped ITRs with A and A′ regions connected with five nucleotides have been found to reduce viral productivity by 75% without affecting genome encapsidation. At the same time, the U-shaped ITRs have provided an increased level of transgene expression in vitro and in vivo [Zhou et al., 2017].
- Codon Optimization: Modifying codons within the ITRs can impact the secondary structure of the RNA and potentially influence viral functions such as packaging and replication.
- Insertion of Regulatory Elements: Incorporating regulatory elements into the ITRs can control aspects of gene expression or transgene stability.
Challenges and Considerations:
- Functional Integrity: While engineering ITRs, it’s crucial to ensure that the essential functions of replication, packaging, and integration are preserved.
- Validation: Engineered ITRs need to be rigorously validated to ensure that the modified vectors retain their desired properties and exhibit consistent behavior across different conditions.
- Regulatory Approval: If ITR engineering is intended for clinical applications, regulatory agencies will require thorough characterization of the modified vectors to ensure safety and efficacy.
ITR engineering is a dynamic field that aims to optimize AAV vectors for various applications. Researchers must balance the desire to enhance vector properties with the need to maintain the essential functions of the ITRs for successful viral replication and gene delivery.
AAVnerGene provide AAV vectors design with different ITR templates.
Zhou et al., 2017: Deletion of the B-B’ and C-C’ regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression
Ling et al., 2016: Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors