Gene Targeting: A Focus on Acute Myeloid Leukemia Gene Silencing and Detection of Methylated DNA by Exploring the Use of Synthetic Nanocarriers and Nucleic Acid Analogs

• Author / Creator

  Ubeda, Anyeld M

• Research in the field of nucleic acids and molecular genetics has enormously enhanced our knowledge about the genetic origins of a myriad of disease. Currently from this knowledge, various gene-targeted therapies and biomarker detection and diagnostic assays are being developed, which are discussed in Chapter 1, as targeting sequences of interest is deemed a simpler task compared to protein targeting. The treatment and understanding of Acute Myeloid Leukemia (AML) development has greatly benefited from the advances in molecular characterization and the World Health Organization (WHO) recognizes certain recurrent genetic abnormalities as diagnosis for the disease. In this thesis, we first demonstrate how we can develop gene silencing therapeutic options targeting messenger RNA (mRNA) in AML cells by delivering small interfering RNAs (siRNA) using polyethylenimine (PEI) carriers. Next, we show how we can target the aberrant formation of DNA adducts in the sequence of the KRAS oncogene using synthetic nucleobase analogs. More specifically, in Chapter 2, we explored the use of RNA silencing as a promising candidate to be used in conjunction with traditional treatments as it has the potential to act in synergy to lower doses, length of treatments and side effects. We identified molecular targets using lipid modified PEI delivery systems that could be used as the basis of therapy during AML progression using the stem-cell like KG1a cell line and primary cells. We identified linoleic acid and lauric acid modified PEI as effective and versatile carriers and showed how they can effectively downregulate the mRNA levels of genes including BCR5 (survivin), and BCL2L12 leading to a reduction in proliferation of the cells. Moreover, we highlight the versality of the synthetic delivery systems and how gene silencing allows for personalized and targeted patient treatment. In Chapter 3, we showed the versatility of incorporating DNA base analogs to oligonucleotide sequences for the detection of O6-methylguanine (O6-meG), a DNA adduct that is highly mutagenic. The base analogs were synthesized by the Sturla group in ETH Zurich to have higher affinity to the methylated guanine in comparison to the non-methylated form. Our main objective was to develop a templated-ligation based detection assay and identify parameters that enhance the selectivity of the synthetic base for downstream sequence amplification. The templated ligation reaction would simplify previously reported detection methods for the same adduct and also give sequence specificity of the location of interest which a lot of previous methods lacked. We examined different parameters of the ligation reaction, modified the reacting probes, and reaction conditions to increase the detection limit of our assay in the presence of non-methylated background DNA. By enhancing the selectivity of the ligation reaction, we aimed to increase the selectivity of the O6-meG detection retaining sequence specificity so that in the future we can design a fast screening test for adduct formation that can be used as a biomarker test for hotspot mutation sites.

• Subjects / Keywords

  • Gene silencing
  • O6-meG detection
  • nucleic acid therapies
  • nucleic acid detection

• Graduation date

  Spring 2019

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-xdgg-kd36

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.