Enhancing the expression of foreign nucleic acids in mammalian cells via synthetic biology

The successful expression of foreign nucleic acids delivered into mammalian is essential to both fundamental research and biotechnology. However, foreign DNAs (e.g., transgenes) integrated into the mammalian genome are subject to epigentic silencing, leading to decreased expression over time. Moreover, synthetic messenger RNAs (mRNAs) are highly immunogenic in mammalian cells, resulting in inflammation activation, adverse immune reactions and reduced translation. The focus of my thesis was to enhance the expression of foreign nucleic acids in mammalian cells through synthetic biology approaches. In the first section (Chapter 2-3), I described a novel platform named SHIELD (Site-specific Heterochromatin Insertion of Elements at Lamina-associated Domains) that I developed for high-throughput screening of barrier-type DNA elements in human cells, by exploiting the naturally occurring heterochromatin spreading inside lamina-associated domains (LADs). SHIELD enables successful insertion of plasmid-sized DNA fragments into LAD loci with high efficiency and fidelity, circumventing the need for tedious clonal isolation. I observed three distinct kinetic classes of transgene silencing and proposed SHIELD could capture transgene repression at mRNA level. Furthermore, following the screening of ~110 DNA elements by SHIELD, I identified two novel CTCF high-affinity binding sequences with barrier activities higher than the widely used cHS4 core region. SHIELD should greatly facilitate the discovery of novel barrier DNA elements from the non-coding genome. In the second section (Chapter 4), I described the development of Z-mRNA as an effective vaccine against SARS-CoV-2. Chemically modified mRNAs hold great potential for therapeutic applications in vivo. Currently, the base modification scheme largely preserves the canonical Watson-Crick base pairing, thus missing one mode of mRNA modulation by altering its secondary structure. In Chapter 4, I described the incorporation of base Z (2-aminoadenine) into mRNA to create Z-mRNA that exhibited improved translational capacity, decreased cytotoxicity, and reduced immunogenicity in mammalian cells compared to unmodified mRNA. In particular, the A-to-Z substitution rendered modified mRNAs less immunogenic than the state-of-the-art base modification N1-methylpseudouridine (m1ψ). As a proof of concept, I developed a Z-mRNA-based vaccine against SARS-CoV-2, which successfully elicited substantial humoral and cellular immune responses in vivo in mice. Z-mRNA expands the current scope of mRNA base modifications towards noncanonical bases, and offers an advantageous platform for mRNA-based therapeutics given its minimal immunogenicity. In the last part (Chapter 5), I summarized my efforts to dissect a large chromosomal domain to identify speckle-targeting sequences in mammalian genome. Genome spatial organization also regulates gene expression, but its underlying mechanism remains largely elusive. My aim was to develop a new strategy to accelerate the dissection of nuclear compartment-associated domains by direct cloning and rewiring of endogenous locus through targeted DNA integration. Such a strategy could contribute to the discovery of DNA sequences that encode information for genome spatial organization.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Zhang, Meng

Contributors dc:contributor

• Zhao, Huimin
• Belmont, Andrew S.
• Schroeder, Charles M.
• Kong, Hyunjoon

• Synthetic Biology
• Genome Engineering
• Mrna Therapeutics

Statement dc:rights

• Copyright 2022 Meng Zhang

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/122082

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F122082