Engineering of viral surfaces and production methods for gene therapy applications
Time: Fri 2026-09-25 09.30
Location: Kollegiesalen, Brinellvägen 8, Stockholm
Video link: https://kth-se.zoom.us/j/67321587061
Language: English
Subject area: Biotechnology
Doctoral student: Hannes Thorell , Proteinteknologi
Opponent: Doktor Bernard Schneider, École Polytechnique Fédérale de Lausanne (EPFL)
Supervisor: Professor Johan Rockberg, Proteinteknologi; Docent Magdalena Malm, Proteinteknologi; Universitetslektor Greta Hultqvist, Dept. of Pharmacy, Uppsala University, Uppsala, Sweden
QC 2026-08-21
Abstract
Several genetic diseases can now be treated using gene therapy. Adeno-associated viruses (AAVs) are widely used in these types of therapies as gene delivery vehicles, where the AAV genome is replaced with a therapeutic gene. However, AAV gene therapies are expensive due to challenging recombinant production. Additionally, AAVs are not organ-specific, increasing the dose required to reach efficacy. This drives up drug prices and risks of side effects, especially towards the liver. This thesis presents methods to improve AAV production and to make AAVs organ-specific.
AAV production requires plenty of plasmid DNA and in drug development phases of AAV-therapies, plasmid purification is highly manual. In the first article, automated plasmid purification is compared to a manual, industry-standard kit. The automated system lowered manual labour by up to 95 %, with similar or up to 14-fold higher AAV titres depending on the AAV production system. Transfection parameters were studied, showing larger transfection complex formation when using automatically purified plasmids.
The next two articles describe the main work of the thesis: the development of a modular platform for organ-selective AAVs. By attaching a type of affinity scaffold protein called Affibody molecules to the viruses, the AAVs became infectious only towards cells displaying the target receptor of the Affibody. The Affibody-AAVs demonstrated selective cell uptake in vitro and modified organ uptake in vivo, avoiding the liver. Cryo-electron microscopy (Cryo-EM) studies of Affibody-AAVs yielded a high-resolution protein structure at 1.67 Å, along with the first structural data of an affinity scaffold integrated into an AAV.
In the final paper, AAV-production was systematically compared between two common AAV production cell lines: adherent HEK293T and suspension HEK293F. HEK293T produced higher AAV titres than HEK293F (3 to 633- fold higher) and viruses secreted from HEK293T up to 316 times more. By creating AAV-hybrids, secretion was increased from HEK293F. Cell expression level studies of heparan sulphate proteoglycans (HSPGs), targeted by the AAV-variant AAV2, showed lowering of HSPG levels may improve AAV2 secretion.
In summary, this thesis describes methods for creating more efficient AAV gene therapies and for improving AAV production. The presented Affibody-AAV platform can also broaden the use of AAV gene therapies, by enabling targeting of AAVs to organs which naturally-occurring AAVs do not reach.