An ITR Faceoff

August 30, 2023 / by Jason Devlin, Ph.D.

AAV ITRs versus Transposon ITRs

Inverted Terminal Repeats (ITRs) are DNA sequences flanking the transgene of interest to signal incorporation into adeno-associated virus (AAV) or genome insertion by transposases. Despite sharing the ITR element name, viral vector and transposon ITRs differ in sequence and important properties that need to be considered in handling these vectors.

The first question to ask is, does your transgene vector contain ITRs? If you’re working with an AAV vector or if you’re working with transposon vectors, the answer is yes for both systems. In this post, I’ll walk through the differences between the two and what that means for your development processes.

Functional significance of ITRs in AAV
While ITRs perform analogous roles in both AAV and transposon vectors, their sequences and properties vary. As the name implies, these sequences are inverted repeats, contain inverted repeats, or are a combination of both. AAV2 ITRs contain a 125 bp sequence in opposite orientations, comprised of 54 bp-long inverted repeats.

The inverted repeat generates a high degree of secondary structure in ITRs, forming a T-shaped hairpin. As AAV are single-stranded DNA viruses, these T-shaped hairpins provide self-annealing templates and Rep protein binding and nicking sites for DNA polymerase-mediated replication of the AAV genome (1,2). While the ITRs of different AAV serotypes differ in sequence, AAV2 Reps are compatible with other serotype genomes and capsids (1).

Transposon ITRs
Unlike AAV, transposon transgene donors are double-stranded DNA templates, so the function of the ITRs is to provide a binding and cutting site for transposases. The inverted nature of transposon ITRs allows for “cut-and-paste” mechanisms of host genome integration ,similar to sticky ends generated in restriction endonuclease-mediated cloning. Here are popular transposon systems used with a description of how their ITRs are arranged:

• Sleeping Beauty
  • Comprised of inner and outer imperfect direct repeats in opposite directions
• piggyBac®
  • Internal 19-bp palindromic sequence and 13-bp terminal sequence with opposite orientations of the terminal sequence on both ITRs
• TcBusterTM
  • Contain a 10-bp sequence of 5’ TNNNNANCAG 3’

Considerations for AAV ITR
Transposase ITRs do not require special attention in the cloning and propagating of transgene vectors, as they are compatible with all standard cell lines and all sequencing approaches. However, the high degree of secondary structure formed by AAV ITRs can pose challenges in working with AAV ITR vectors. Deletions in the ITRs, difficulties with sequencing the ITRs via Sanger sequencing, and poor manufacturing yields are well-documented but poorly understood.

Problematic ITR-bearing plasmids
The challenges associated with AAV ITR-bearing plasmids is not that they have ITRs, but that the AAV ITRs themselves are palindromic and form a high degree of secondary structure. Therefore, checking for AhdI and SmaI sites in the ITRs is a good indicator that the vector has AAV ITRs, requiring special considerations.

The European Molecular Biology Open Software Suite (EMBOSS) has web-accessible tools for finding palindromes and inverted repeats, called palindrome and inverted, respectively. Submitting a single AAV ITR results in long (>40 bp) and high-scoring palindromes and inverted repeats, whereas screening single transposon ITRs results in shorter palindromes and repeats.

DNA secondary structure predictors can also be used to visualize the palindromic nature or lack of palindromic nature of ITRs. AAV ITRs are predicted to form structures with a few long and perfectly complimentary hairpins, whereas transposon ITRs have comparatively shorter hairpins with mismatches or gaps.

ITR stability solutions
Aldevron’s solutions for constructing and propagating AAV vectors include the Nanoplasmid® vector and REVIVER® host strain. The Nanoplasmid vector utilizes an R6K replication origin rather than a conventional pUC-based origin, resulting in better performance with structured DNA-containing vectors. The REVIVER cell line was engineered to produce constructs with difficult long inverted repeats with outstanding manufacturing yields and stability.

In our experience, verifying AAV ITR stability through restriction enzyme digestion of SmaI (or XmaI) 5’ CCCGGG 3’, and AhdI (5’ GACNNNNNGTC 3’) can aid in screening of ITR integrity. Furthermore, sequencing within and through AAV ITRs can be done not only by Illumina-based sequencing but also by Nanopore sequencing.

The Takeaway
Advances in plasmid backbones, cell strain engineering, and sequencing technologies have improved the fidelity, production, and screening of AAV plasmids. Knowing the properties of the ITRs is critical to ensure that you have the right production capabilities and quality control level for your transgene vector of interest.