Design and delivery of genes encoding oncotoxic proteins for cancer therapy

Development of cancer depends on mutations within the organisms’ own cells. Mutations
of random loci within the genome, affecting a wide variety of genes might lead to cancer.
The resulting versatility of the disease is one of the main reasons, despite great progress
was made, that many cancers still cannot be cured, and cancer is still one of the leading causes
of death worldwide. Conventional therapies, like surgery, radio- and chemotherapy still are the
fundament of most treatment strategies up to date. However, their success is limited and often
associated with severe side-effects. For the same reason, future treatment strategies are shifting
more and more in the direction of customized treatments, adapted to the genetic profiles of cancer
cell populations of individual patients. Customized, more specific treatment strategies, which
selectively target cancer cells, tend to be more effective while having less side effects.
The present thesis aimed to combine the potential use of an anticancer gene, which is toxic
exclusively to certain types of cancer cells, with a targeted gene delivery strategy, aiming to
deliver the therapeutic gene selectively to target cells. Both, gene therapeutics and drug targeting
potentially allow a patient- specific treatments and have an advanced security profile compared
to unspecific drugs. During and for the developmental steps of a suitable gene delivery strategy
carried out in the present thesis, zebrafish embryo-based models were developed, validated and
used.
The anticancer gene which was focused on during this thesis expresses the nonstructural protein
1 (NS1) from parvovirus H1 (H1-PV). H1-PV is toxic exclusively to certain types of cancer cells
and has therefore reached clinical trials. We could demonstrate that the NS1 gene can be used as
cancer therapeutic without any additional viral compounds. Furthermore, a predictive marker
for NS1 responsiveness could be identified, which is important for a customized therapeutic
approach. In a next step, mutants of NS1 were rationally designed and screened for the most
potent candidate. This led to the identification of NS1 mutant NS1-T585E, which showed a 30%
increased cytotoxicity compared to NS1-wt, while retaining its specificity. The present work also
contributes to deepening the understanding of how NS1 interacts with its host-cells and about its
cancer cell selectivity. Altogether, the combined work on NS1 will pave the way for its use as a
future anticancer therapeutic.
After optimization of the anticancer gene NS1, various gene- and other drug-delivery and -
targeting strategies were explored. For many of these steps, the use of zebrafish embryos as
screening tools was evaluated. This included the generation of zebrafish embryo-based antibiotic
drug screening models, which allow efficiency testing as well as pharmacological and mechanistic
insights on drug effects and bacterial infections. Zebrafish embryos were also used for the assessment
of biodistribution and blood clearance of a variety of drug carriers. For drug-targeting
experiments, further sophisticated models and protocols were developed, which will facilitate
future studies in the field.
Using zebrafish embryo-based models, this work also contributed to the development of a feasible
and scalable production method of extracellular vesicles, which was a major burden to their
use for drug delivery alternatively to synthetic particles so far. Zebrafish embryo-based models
were also used to assess the feasibility of peptide MyrB mediated targeted delivery of enzymes
to hepatocytes. This work further proved the translatability of zebrafish-based experiments to
higher vertebrates.
The most promising vectors for the in-vivo delivery of therapeutic DNA plasmids so far appeared
to be lipid nanoparticles (LNPs). Long circulating lipid nanoparticles were designed during this
work, and we demonstrated their capability to effectively and selectively deliver DNA using
passive targeting to solid tumors in mouse xenografts after i.v. administration.
Active cancer targeting was achieved by coupling the monoclonal anti-HER2 antibody Herceptin
(Her) to long-circulating LNPs. The produced Her-LNPs showed the capability to selectively
transfect HER2 overexpressing cells with almost zero cytotoxicity and a high transfection efficiency
in-vitro. It was also demonstrated that Her-LNPs keep their capability to selectively bind
and transfect HER2 positive cells in-vivo, using zebrafish embryo xenografts.