Nano- and micro-scale polymeric conjugate systems for editing and detection of gene targets

Abstract

Advances in molecular biology techniques, absorbing the realm of biochemistry and genetics, have dramatically improved the understanding of the molecular mechanisms of human diseases. The development of innovative strategies for target-specific treatment and diagnostics has also followed, utilizing various nano- and micro-particles with unique properties that target the disease biomarkers. Herein, I present platform technologies using nano- and micro-scale polymer conjugates for the editing of target genes as a therapeutic, as well as the detection of pathogen biomarkers for rapid diagnostics. This paper is organized as follows. The clustered regularly interspaced short palindromic repeats (CRISPR) and its CRISPR-associated proteins (Cas9) have recently been well established as a versatile gene-editing tool, which offers a significant advantage in target-specific gene editing due to simplicity in design and procedures. Even though viral delivery has been the most widely utilized due to the benefits of high transfection efficiency, it has many limitations for applications in the clinic. In chapter 2, I introduce a non-viral delivery method based on a polymer derivatized CRISPR-complex (Cr-Nanocomplex), using sgRNA targeting antibiotic resistance. Recombinant Streptococcus Pyogenes Cas9 (SpCas9) was directly conjugated by a covalent bond with a branched polyethyleneimine, a cationic polymer, as a carrier for packaging and transferring sgRNA to bacteria, showing maintenance of Cas9 endonuclease activity after the chemical modification of protein. I hypothesized that the covalent incorporation of a minimal amount of carrier material into each molecule of Cas9 would enable efficient delivery to bacteria, which provides advantages over the conventional non-covalent lipid-based formulations. The Cr-Nanocomplex targeting mecA – the target gene involved in methicillin resistance – could be successfully delivered into Methicillin-resistant Staphylococcus aureus (MRSA), which have been known hard to transfect due to the thick bacterial cell wall. I also demonstrated that the delivered Cr-Nanocomplex can suppress the growth of MRSA by enhanced efficiency in editing of the bacterial genome, resulting in target-specific killing of the multidrug-resistant bacteria. The Cr-Nanocomplex was further applied by incorporation of donor DNA for the intracellular delivery and precise gene editing in mammalian cells in Chapter 3. To evaluate the gene-editing efficiency, monoclonal cell lines of a fluorescent reporter system were used, and the Cr-Nanocomplex was applied to induce precise gene-editing including homology-directed repair (HDR) as well as non-homologous end joining (NHEJ). The delivery of the Cr-Nanocomplex into various mammalian cells resulted in greatly increased intranuclear uptake of both Cas9 and sgRNA, compared to the native complex. I also demonstrated the feasibility of the Cr-Nanocomplex on its efficacy in precise gene-editing. According to these results, the Cr-Nanocomplex can be potentially used in vivo and is expected to show efficient intracellular delivery and gene-editing efficacy, while minimizing toxicity problems. For immediate and proper treatment of a disease, a rapid and simple assay that allows target-specific detection is essential. In Chapter 4, I proposed a facile molecular diagnostic platform suitable for point-of-care testing (POCT) using the coffee ring effect for detecting antibiotic resistance in bacteria. The assay solution including polymeric microbead-probe conjugates and isothermally amplified nucleic acid targets is applied on a tensile surface, and the spatial pattern is observed. The presence of the nucleic acid target resulted in suppression of the coffee ring effect. In this thesis, I developed the Cr-Nanocomplex and applied the system to induce efficient gene editing in both prokaryotes and eukaryotes. I also developed a diagnostic platform that allows point-of-care testing and can be utilized for treatment decision making and monitoring treatment efficacy. Intuitive diagnostic methods have been proposed, and practical applicability has been demonstrated for a specific target. These studies have allowed me to obtain a broad and in-depth understanding of specific gene targets related to human diseases, and the ability to apply different biotechnology approaches to target the genes for medical translation.