Abstract
Baculoviruses (BVs) represent promising viral vectors for gene therapy and have an unrivalledDNA cargo capacity. More recently, the Berger group demonstrated that BVs can also co-deliver
customizable, heterologous protein cargos to human target cells by engineering protein-loaded
baculoviruses or pBVs. pBVs exploit the passive diffusion of cytoplasmic proteins into BVs during
virus generation in insect cells. By use of an inducible protein heterodimerization system,
selected protein cargos can also be actively loaded into selective pBVs or spBVs. This thesis
focuses on optimizing BVs as DNA delivery and gene therapy vectors by harnessing this protein
delivery functionality. The first part of the thesis predominantly explores the use of spBVs for
CRISPR-mediated genome engineering. spBVs were engineered to deliver Cas9 protein (Cas9-
spBVs), which demonstrated successful multiplexed genome engineering in a range of human
cells. Intriguingly, Cas9-spBVs also afforded safer editing than plasmid transfection approaches
and thus provide a valuable alternative to currently available delivery systems. Following on from
this, other protein cargos were investigated to optimize application of (s)pBVs in gene therapy.
One limitation of BVs is their silencing by mammalian innate immune responses. To tackle this,
pBVs were engineered that harbored immunomodulatory proteins to disrupt these pathways.
Compelling evidence that innate immune pathways, notably cGAS/STING signaling, can be
targeted to increase BV transgene expression, is presented. However, the potential of
immunomodulatory-pBVs remains unclear and requires further exploration. To strengthen the
experimental setups, a qPCR approach for assessing transgene loss from recombinant BV
genomes is also described and validated. In summary, this thesis presents a detailed exploration
of the protein delivery capacity of BVs. We envision further development of (p)BVs for
therapeutic genomic interventions that leverage the unique DNA cargo capacity advantage of BVs.
Lastly, when research activity was curtailed due to COVID-19, a passive immunization reagent to
neutralize SARS-CoV-2 was developed and this project included in a standalone Chapter in the
thesis.
| Date of Award | 10 Dec 2024 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Supervisor | Imre Berger (Supervisor), Christiane H Berger-Schaffitzel (Supervisor) & Mark D Szczelkun (Supervisor) |