University of Jyväskylä

Dissertation: 17.6.2017: Hepatitis E to benefit breast cancer treatment in the future

Start date: Jun 17, 2017 12:00 PM

End date: Jun 17, 2017 03:00 PM

Location: Ylistönrinne, YAA303

M.Sc. Marie Stark defends her doctoral dissertation in Cell and Molecular Biology ”Recombinant Nanocapsids for Targeted Theranostic Delivery”. Opponent  Professor John Johnson Jr. (Scripps Research Institut, California) and custos Professor Varpu Marjomäki (University of Jyväskylä).

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Marie Stark
Diseases such as cancer and genetic disorders can be difficult to detect and even harder to treat because there are no foreign pathogenic targets. Instead, an individual’s dysfunctional cells and cell functions of the are the primary disease targets for detection and treatment. Difference in the disease expression between individuals can also make standard diagnostic tools and therapies much less reliable and ineffective. This has driven disease detection and management towards methods of molecular-level targeting. Targeted delivery seeks out diseased cells for diagnosis and/or treatment, also known as theranostics. Targeting theranostics to diseased tissues improves their specificity, reducing therapeutic dosage and minimizing side effects.  Nanosized particles, or nanoparticles are used as stable carriers of theranostics such as fluorescent or drug molecules. These theranostic nanoparticles serve as nano-sized platforms to deliver detection and/or therapeutic molecules to diseased tissues, without effecting healthy tissue. Viruses are naturally occurring nano-sized platforms that are adapted for stable, targeted delivery of virus material to specific tissues, causing viral infection in those infected tissues. While viral infection is not desirable, it is possible to design virus-like particles that have the tissue-targeting and stable delivery capacity of viruses, without carrying the components necessary for virus infection. In addition, the study of virus delivery mechanisms can offer insight in to theranostic design.

Marie Stark’s thesis outlines several applications of recombinant virus and virus-derived supramolecular complexes for both design and investigation in to targeted delivery. Under the supervision of Adjunct Professor Varpu Marjomaki, Professor Hannu Hakkinen, and Professor R Holland Cheng, Marie is defending her PhD thesis on June 17th 2017. Marie’s thesis sought to develop and modify a virus-based theranostic nanoparticle for targeted delivery. Virus-like particles derived from Hepatitis E Virus capsid protein, forming empty icosahedral nanocapsids, were produced in R Holland Cheng’s lab at UC Davis to serve as a platform to carry vaccines and/or drugs into the body. With Hannu Hakkinen’s expertise, gold nano-clusters were directly bound to the HEV nanocapsids to optimize the detection and structural determination of modified nanocapsids. To better understand virus delivery mechanisms, Marie studied recombinant enterovirus infection in Varpu Marjomaki’s lab.

Stark’s thesis work involved both genetic and chemical modification of virus like particles to bind both cancer targeting molecules and gold nanocluster detection molecules to the surface of HEV nanocapsids. Specific tumor targeting was successful in rodent studies with chemically modified nanocapsids. Gold nanoclusters were also bound to the surface of chemically modified HEV nanocapsids and imaged in cryo TEM from which a 3D density model of gold-bound nanocapsid was resolved using 3D reconstruction. Lastly, recombinant enterovirus B studies carried out in Varpu Marjomaki’s lab suggested that the virus’s targeted specificity for a protein expressed on the surface of cells is primarily responsible for virus life-cycle despite other components of virus infection.

Abstract

Developments in diagnostic and therapeutic delivery are trending towards molecular level targeting with nano-platforms. Targeted delivery reduces generalized distribution by localizing diagnostic and/or therapeutic (theranostic) molecules to an intended target site. The first section of this thesis proposes Hepatitis E Virus (HEV) nanocapsids as a vector to stabilize and target theranostic delivery. Derived from the capsid protein of HEV, a fecoorally transmitted virus, HEV-like particles self-assemble in to non-infectious, nanocapsids that can withstand harsh protease and pH conditions in the mucosal system. The flexible nanocapsid surface protrusion domain is amenable to substantial modification. Chemical modulation of nanocapsids was achieved through surface conjugation to single solvent exposed cysteine sites. In this thesis, nanocapsids chemically modulated with tumor-targeting ligands exhibit cancer cell-specific binding and internalization, as well as in vivo  tumor detection. We also used cysteine sites to conjugate thiolate-protected gold nanoclusters (AuNCs), which have molecule like qualities distinct from colloidal gold nanoparticles (AuNPs). Specifically, Au102 (p MBA)44  (Au102 ) and maleimide-linked Au102  (Au102 _C6 MI) were conjugated to nanocapsid cysteine through place exchange and maleimide-thiol coupling, respectively. Au102 _C6 MI-bound nanocapsids were imaged in cryoEM and a 3D structure was determined. The resolved structure of AuNC-bound nanocapsid revealed a 5-fold ring density attributable to AuNC densities. Rigid modelling supported this finding. In the last section of this thesis, a recombinant enterovirus was engineered to distinguish the role of structural vs. non-structural proteins in Enterovirus B infection kinetics, replication and infection persistency. The results indicated cell-receptor binding likely triggered lytic vs. non-lytic infection, providing insight in to adaptive mechanisms of native virus cell delivery.

Keywords: Bioconjugation; Gold nanocluster

More information

Marie Stark
marie.c.stark@jyu.fi