title: The hidden syntax of AAV: Insights into epigenetic regulation and discovery of a dinucleotide pattern in Adeno-associated Virus (AAV)
creator: Baumgartl, Conradin Franz Daniel
subject: ddc-500
subject: 500 Natural sciences and mathematics
subject: ddc-570
subject: 570 Life sciences
subject: ddc-600
subject: 600 Technology (Applied sciences)
description: Recombinant Adeno-associated Virus (rAAV) is an auspicious gene-delivery vector for human gene therapy. Even though there are multiple approved rAAV-based drugs available, many underlying molecular mechanisms remain uncertain. This thesis is dedicated to the investigation of two related areas of AAV's biology: (i) investigation of epigenetic regulation of delivered DNA comparing two distinct AAV vector capsids, and (ii) exploration of a sequence pattern conserved for the genus Dependoparvovirus, which may contribute to improved transgene cassette design.    AAV-delivered DNA mainly persists as circular episomal DNA within the nuclei of transduced cells. The extent of epigenetic regulation that the transduced cells enact on the delivered vector DNA and the influence of the capsid are severely understudied. In a long-term mouse model experiment, I found that the DNA methylation in the formed episome is negligible using nanopore sequencing. However, histone modifications were differentially deposited on AAV2- and AAV9-delivered episomes. At later timepoints, expression-beneficial histone acetylations on the AAV9-delivered transgene became lost, and its expression was repressed. AAV2 appeared to have an expression-permissive regulation, but was being held back by its unpacking speed.    In the second part of this work, I describe the discovery of a pronounced 15 bp YY/RR dinucleotide pattern unique to the genus Dependoparvovirus. It resembles the 10 bp histone-positioning code in eukaryotic genomes, but the longer repeat length likely enables a different binding partner, potentially those involved in viral replication or packaging. The biological importance of this pattern was demonstrated through its enrichment during the selection of shuffled AAV cap sequences. Additionally, I could show that rAAV vectors that contained sequences with the wild-type periodicity produced higher titers in a small-scale production system, suggesting a functional role necessary for vector production. Based on the evidence provided, an increased interaction of the DNA with Rep proteins is a likely explanation for the presence of this pattern. The described pattern could be harnessed in the future to improve rAAV transgene cassette designs.
date: 2025
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserver/36959/1/2025_PhD_Conradin_Baumgartl.pdf
identifier: DOI:10.11588/heidok.00036959
identifier: urn:nbn:de:bsz:16-heidok-369598
identifier:   Baumgartl, Conradin Franz Daniel  (2025) The hidden syntax of AAV: Insights into epigenetic regulation and discovery of a dinucleotide pattern in Adeno-associated Virus (AAV).  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/36959/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng