SiRNA Therapy with Lipid-modified Polymers in Chronic Myeloid Leukemia

• Author / Creator

  Valencia Serna, Juliana

• BCR-ABL fusion oncogene is the main driver mutation of Chronic Myeloid Leukemia (CML) that controls initiation, maintenance and progression to more aggressive stages of the disease. Current therapies with tyrosine kinase inhibitors in CML involve certain limitations, such as drug resistance and insensitivity. Therefore, RNA interference (RNAi) represents a promising tool for the silencing of over-expressed genes that can regulate unwanted biological effects produced in diseases such as cancer. CML arises from the acquisition of the BCR-ABL oncogene in normal hematopoietic stem cells that progressively outgrow the normal hematopoietic stem cells in the stem cell niche and subsequently leads to an uncontrolled expansion of immature myeloid cells. Tyrosine kinase inhibitors (TKIs) used to target the ABL tyrosine kinase have shown significantly increased clinical outcomes in CML patients, however, patients in blast crisis phase are more likely to relapse and develop resistance often due to mutations in the TKI domain and innate or acquired insensitivity of CML stem cells. RNAi represents a promising alternative for the treatment of CML as small interference RNA (siRNA) molecules can be applied to the silencing of specific targets that modulate the biological outcome and induce therapeutic effects. However, potent carriers that can overcome delivery barriers of RNAi agents and are effective in difficult-to-transfect cells, such as CML cells, are needed for the progression of siRNA-based therapies towards clinical applications. In this thesis work, we explored the use of lipid-modified polyethylenimines (PEI) of low molecular weights (0.6, 1.2, 2.0 kDa) in in vitro and in vivo CML models and evaluated their siRNA transfection efficiency in terms of cytotoxicity, siRNA uptake, internalization, gene silencing and biological effects (i.e, apoptosis, cell growth, and cell colony growth). siRNA transfection efficiency of lipid-modified polymers was evaluated in CML K562 cell lines grown in suspension and adhering to a fibronectin (FN) surface, in an in vivo CML (K562) model, and in CML patient cells. The hypothesis of this thesis is that lipid-modified polymers will facilitate siRNA delivery to CML cells efficiently to obtain functional outcomes that could be potentially used as therapy for CML. Among the lipid substituents evaluated for PEI modification, palmitic acid (PA), alpha linoleic acid (LA), and cholesterol (Chol) have proven to be highly efficient in delivering siRNA and silencing of the green fluorescent protein (GFP)- reporter gene in K562 cells grown in vitro in suspension (PA, LA, and Chol) and on K562 cells grown adhering to FN-modified surfaces (LA). Moreover, BCR-ABL-siRNA transfection produced a significant decrease of BCR-ABL mRNA in K562 cells (PA, LA, and Chol) and CML primary cells (LA, and Chol) which subsequently resulted in significant increase of apoptotic cells, cell growth inhibition, and reduced ability to form cell colonies in vitro in comparison with control groups. Although a decrease in BCR-ABL mRNA was not evident in CML models in vivo, a retardation in the tumor growth was observed in comparison with control groups. The results from this study revealed the potential of siRNA-based drugs and are encouraging for the future design of non-viral delivery system with clinical translation capabilities for the treatment of CML. This thesis work provides ample opportunities for delivery systems that could be useful to silence other target genes in CML and other leukemias (i.e., acute lymphoblastic leukemia and acute myeloid leukemia) for therapeutic purposes.

• Subjects / Keywords

  • Cationic polymer
  • Therapy
  • Chronic Myeloid Leukemia
  • siRNA
  • Leukemia

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3ZS2KW0X

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.