Inverted Terminal Repeats (ITRs) are short DNA sequences found at the ends of the genome of parvoviruses, most notably the Adeno-Associated Virus (AAV). These sequences act as the minimal genetic signal required for the viral genome to be handled and packaged. The distinct nature of ITRs allows scientists to repurpose the AAV shell into a non-replicating delivery vehicle, transforming them into foundational tools for modern gene therapy. By understanding these sequences, researchers can efficiently deliver corrective genetic material to human cells to treat inherited and acquired diseases.
The Unique Molecular Structure of ITRs
ITRs are palindromic, meaning the sequence of nucleotides reads the same backward on one strand as it does forward on the complementary strand. The sequence at one end of the viral genome is an inverted complement of the sequence at the other end. This internal complementarity allows the single-stranded DNA of the AAV genome to fold back upon itself immediately upon entering a cell.
The sequence forms a stable, double-stranded structure resembling a hairpin or a “T” shape. Wild-type AAV serotype 2 (AAV2) ITRs are typically composed of 145 base pairs and feature distinct regions labeled A, B, and C, with their corresponding inverted complements A’, B’, and C’.
This T-shaped structure protects the genome’s ends and is remarkably stable. The stability of this hairpin loop makes it a recognizable docking site for viral and host cell machinery.
Biological Role in Viral Life Cycles
In the natural life cycle of the AAV, ITRs serve a dual function that guides viral replication and assembly. The most immediate function of the ITR hairpin is to act as a self-priming mechanism for DNA replication. Because the folded ITR creates a double-stranded section, it provides the free 3′ hydroxyl group necessary for host polymerases to begin copying the viral genome without needing a separate RNA primer.
The ITR sequence also contains specific recognition sites that are bound by the AAV replication proteins, known as Rep proteins. These sites include the Rep Binding Element (RBE) and the Terminal Resolution Site (trs). The Rep proteins bind to the RBE and then cleave the DNA at the trs, initiating the process of unwinding and synthesizing a new DNA strand.
Beyond replication, the ITR acts as the essential packaging signal for the viral capsid. The Rep proteins, having recognized the ITR, guide the newly formed viral DNA into the pre-assembled protein shell. Only DNA flanked by these ITR sequences is efficiently recognized and packaged, ensuring that only the correct genetic material is enclosed within the mature virion.
ITRs as the Engine of Gene Therapy Vectors
The utility of ITRs in gene therapy stems from their ability to be the sole viral components required to facilitate packaging and delivery. Scientists engineer a recombinant AAV (rAAV) vector by removing the native viral genes, rep and cap, which encode the replication and capsid proteins. These removed genes are replaced with a therapeutic gene cassette containing the corrective gene sequence.
The ITRs are intentionally retained and placed on either side of this new therapeutic gene cassette, acting as the new bookends for the artificial genome. Because the ITRs are cis-acting elements—meaning they must be located on the same DNA molecule—they ensure that only the therapeutic DNA is recognized and inserted into the AAV capsid. The viral proteins needed for packaging are supplied separately on helper plasmids, creating a non-replicating, therapeutic particle.
Once the rAAV vector enters a human cell, the ITRs facilitate the conversion of the single-stranded DNA into a stable, double-stranded form. This double-stranded form can persist as an episome in the nucleus, allowing for long-term expression of the therapeutic protein.
Current Challenges in ITR-Based Gene Delivery
ITR-based gene delivery systems face practical limitations that researchers are actively working to overcome. These challenges primarily relate to the physical constraints of the vector and the complexity of manufacturing.
Limited Payload Capacity
One significant constraint is the limited payload capacity of the AAV capsid. The space between the two ITRs can accommodate a therapeutic gene cassette of approximately 4.7 kilobases (kb). This size restriction means that therapeutic genes larger than this limit cannot be delivered in a single vector. To address this, scientists are exploring methods like “dual-vector” delivery, where the gene is split and delivered by two separate AAVs that recombine inside the target cell.
Production Instability
A further challenge lies in the difficulty associated with producing high-quality, clinical-grade vectors at the necessary scale. The complex secondary structure of the ITR, including its high Guanine-Cytosine (GC) content, can cause instability in the production plasmids used to manufacture the vectors. This instability can lead to deletions or truncations in the ITR sequence, potentially reducing the efficiency of vector packaging.
Ongoing research efforts focus on modifying the ITR sequence to enhance its performance. Approaches include creating hybrid ITRs from different AAV serotypes or developing ITRs with reduced immune-stimulating CpG motifs to improve stability and reduce unwanted host immune responses.