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primary topic: Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors

Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors
type: Article
abstract: Gene therapy using adeno-associated virus (AAV)-based vectors has advanced significantly in recent years, with multiple approved therapeutics now available on the market. Engineering of the capsid, transgene, and promoters has shown immense potential to improve the safety and efficacy of these drugs. The only vector component that has mostly remained in its original state is the viral replication origin, the inverted terminal repeats (ITRs). Several ITR variants have so far shown potential to improve vector efficacy and safety as well, implying that the parallel screening of large ITR libraries could improve the identification of superior variants. The reason this has never been attempted for variants of the central part of the ITR may be the strong secondary structure of the ITRs, severely impairing the cloning of novel variants. Additionally, the partial loss of the ITRs due to processing by the host’s DNA repair machinery prohibits tracing of the ITR variants upon transduction. To overcome these bottlenecks, a rarely used plasmid design was harnessed in the present work to facilitate the cloning of more than 110 newly designed ITR variants. This was complemented with a novel Sanger sequencing based method for fast and reliable ITR sequence verification in plasmid DNA, which proved effective for all conventional and most alternative ITR structures. To trace the ITR variants during production and transduction, they were tagged with a barcode sequence in the transgene of the vector genome. Intriguingly, after vector production with a pool of ITR variants, the barcoding also enabled the identification of a novel ITR repair pathway that relies on the presence of ITR sequences as repair templates in trans. This ITR trans-repair was detectable in all parts of the ITR and impedes the barcode-ITR association in pooled productions. Importantly, though, this repair could be circumvented by separate production of each ITR variant, which also facilitated the maintenance of hi…
date: 2025
author list: authors
type: EPrint
type: DoctoralThesisEPrint
is part of: repository
has document: Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors (Other)
has document: Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors (PDF)
subject: 570 Biowissenschaften, Biologie

Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors (PDF)
type: Document
has file: PhD Thesis Felix Bubeck final HQ.pdf
has part: PhD Thesis Felix Bubeck final HQ.pdf

Felix Bubeck
type: Person
given name: Felix
family name: Bubeck

Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors (Other)
type: Document
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Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors (Other)
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Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors (Other)
type: Document
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Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors (Other)
type: Document
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Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors (Other)
type: Document
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primary topic: Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors

570 Biowissenschaften, Biologie
type: Concept
preferred label: 570 Life sciences @en