The Inverted Terminal Repeats (ITRs) in adeno-associated virus (AAV) vectors are essential for the replication, packaging, and integration of the viral genome. Ensuring the stability of ITRs is crucial for the functionality and effectiveness of AAV vectors.
ITR instability
The challenges associated with the instability of ITRs in AAV plasmids are well recognized in the field of molecular biology and vector design. The structural properties of ITRs, including their palindromic nature and high GC content, make them susceptible to mutations and deletions during plasmid propagation in bacteria. This instability can have significant implications for AAV vector production and downstream experiments.
ITR Instability in AAV Plasmids: The palindromic nature and high GC content of ITRs make them prone to deletions during bacterial propagation. The 18 G (or C) tail immediately outside the ITRs in certain plasmid backbone has been shown to exert an additional destabilizing effect that can be addressed by the removal of these sequences[Wilmott et al., 2019]. Mutations can lead to full or partial deletions of ITR sequences in the plasmids.
Population Variability: Even with best practices, AAV plasmid preparations can contain a significant fraction (5-15%) of plasmid DNA with mutated ITR sequences.
Consequences of ITR Disruption: Disruption of ITRs can lead to reduced efficiency in viral packaging and increased variability in downstream experiments.
Detecting ITR Lost or Mutations
Due to the frequency of truncations in AAV plasmids, continuous monitoring of ITR integrity is essential, especially prior to viral packaging.
Restriction Digests
- SmaI, XmaI or SrfI: There are commonly used for screening ITR deletions, which target the C-C’ arm of the ITR sequence, which can provide insight into potential deletions.
- MscI: It targets the terminal resolution site of ITR.
- AhdI: It targets Rep binding element at B-domain (RBE’).
Traditional restriction digests are visualized through gel electrophoresis and can be use to detect ITR lost. The limited resolution of gel electrophoresis makes it challenging to detect subtle changes such as small deletions or point mutations that occur outside the restriction sites.
Whole plasmid sequencing
Whole plasmid sequencing enables the complete sequencing of a plasmid DNA molecule using advanced long-read sequencing technologies. Unlike traditional sequencing methods that may require the use of primers, whole plasmid sequencing provides accurate and efficient results without the need for these additional DNA fragments.
Here’s how the process works:
Long-Read Sequencing Technology: Whole plasmid sequencing utilizes advanced sequencing platforms that are capable of generating long sequence reads. These technologies allow for the sequencing of longer DNA fragments in a single read compared to traditional short-read sequencing methods.
Library Preparation: The plasmid DNA is prepared into a sequencing library. This involves fragmenting the DNA and attaching special adapters to the ends of the fragments. These adapters enable the DNA to be loaded onto the sequencing platform.
Sequencing: The prepared library is then loaded onto the long-read sequencing platform. The technology reads the DNA fragments in long stretches, generating sequence reads that can span the entire length of the plasmid without the need for additional primers.
Assembly and Analysis: The long sequence reads are analyzed using bioinformatics tools to assemble the DNA fragments into the complete plasmid sequence. This assembly process is facilitated by the longer read lengths, which can help resolve repetitive or complex regions in the plasmid.
The benefits of whole plasmid sequencing for AAV plasmids with long-read technologies include:
Accuracy: Long-read sequencing technologies provide high accuracy, reducing errors and increasing confidence in the obtained sequence.
Efficiency: Sequencing the entire plasmid in a single read eliminates the need for additional PCR amplification and primer-dependent methods, streamlining the process.
Detection of Structural Variations: Long-read sequencing can reveal structural variations, such as insertions, deletions, or rearrangements, that might be missed by shorter read lengths.
Complex Regions: Whole plasmid sequencing is particularly useful for plasmids with repetitive or challenging regions, such as ITR, that are difficult to resolve using short-read technologies.
Overall, whole plasmid sequencing with long-read technologies offers a powerful and efficient approach to accurately determine the complete sequence of AAV plasmids, providing researchers with valuable insights into plasmid genetics, functions, and applications. AAVnerGene provides whole plasmid sequencing for AAV plasmids on request.
ITR sanger Sequencing
Sanger sequencing is widely recognized as the gold standard for confirming plasmid sequences, providing accurate and detailed sequence information. However, it faces difficulties in reading through ITR regions. The strong secondary structure of ITRs, which is due to their palindromic and repetitive nature, presents a challenge to DNA polymerization during sequencing. Strategies commonly used to enhance sequencing of difficult templates, such as the addition of DMSO and/or betaine, do not effectively overcome the challenges posed by ITR secondary structure.
Some companies has developed a novel Sanger sequencing method that allows qualitative assessment of the integrity of these ITR regions. AAVnerGene collaborates with those companies to offer AAV ITR Sanger Sequencing services.
Methods to prevent ITR lost or mutation
Use of recombination-deficient bacterial strains
Recombination-deficient Escherichia coli strains such as NEB stable (New England Biolabs, cat. no. C3040H), Stbl3 (Invitrogen, cat. no. C737303), or SURE 2 competent cells (Agilent, cat. no. 200152) are commonly used for cloning AAV constructs to reduce the likelihood of ITR deletions during plasmid propagation. At AAVnerGene, we use NEB stable strains to produce all AAV plasmids.
Selection of Smaller Bacterial Colonies
While not universally effective, selection of smaller bacterial colonies from agar plates reduces the probability of choosing clones with ITR deletions, as ITR loss can confer a growth advantage on host bacteria[Wilmott et al., 2019].
Minimization of Bacterial Culture Times:
It’s recommended to keep bacterial culture times relatively short, around 14-16 hours at temperatures of 37°C[Wilmott et al., 2019]. This practice aims to reduce the chances of mutations or changes occurring during bacterial growth. Longer culture times might increase the likelihood of mutations, which could impact the quality of the AAV plasmid.
Check ITR integrity frequently
Analyzing the integrity of ITRs after each DNA preparation is a critical quality control step when working with AAV plasmids. This practice helps ensure the consistency and reliability of your AAV vectors throughout your research or applications.
Use engineering ITRs
ITR with terminal detection(ITR130)
It has been demonstrated that different deletion at terminal of ITRs ( 11-15 bp) do not affect viability and could be used in AAV production[Shitik et al., 2023]. It is rapidly repaired during following second-strand DNA synthesis.
ITR with 11 bp deletion in C-domain(ITR119)
This mutation has been observed during AAV plasmid preparation. This deletion would not significantly affect AAV package and can be corrected during AAV production[Samulski et al., 1982] [Savy et al., 2017][Tran et al., 2020][Rebecca et al., 2019}.
CpG-free ITR
Replacing the wild-type ITR with the CpG-free ITR did not affect vector genome encapsidation. However, the vector yield was decreased by approximately 3-fold due to reduced vector genome replication[Pan et al., 2022]. The CpG-free ITRs were demonstrated to be stable in bacterial passaging.
At AAVnerGene, we provide different versions of AAV ITRs on request. Both ITR130 and ITR119 can be
References
AAV- based vector improvements unrelated to capsid protein modification. Front Med (Lausanne). 2023 Feb 3;10:1106085.
Rational engineering of a functional CpG-free ITR for AAV gene therapy. Gene Ther. 2022 Jun;29(6):333-345.
AAV genome modification for efficient AAV production. Heliyon. 2023 Apr 1;9(4):e15071.
A User’s Guide to the Inverted Terminal Repeats of Adeno-Associated Virus. Hum Gene Ther Methods. 2019 Dec;30(6):206-213.