Lipopolymer Mediated siRNA Therapy for Cancer: Focus on Acute Myeloid Leukemia

  • Author / Creator
    Landry, Breanne K
  • Protein silencing by small interfering RNA (siRNA) is a promising treatment strategy for cancer as over-expression of proteins is largely responsible for cancer cells’ infinite proliferation, evasion of cell death and multi-drug resistance. However, siRNAs require a carrier as their biological instability, negative charge and large molecular weight prevent cellular delivery. In this thesis, I first provide a review of current non-viral siRNA carrier strategies designed to protect and deliver the siRNA to the cell cytoplasm for RNAi activity and then follow with an over-view of the current state of siRNA development with non-viral carriers specifically in leukemia. One promising cationic polymer for siRNA delivery is high molecular weight polyethylenimine (PEI); however, its toxicity is an obstacle for clinical use. This thesis investigates a library of low-molecular weight (2 kDa) PEI with hydrophobic (lipid) modifications as siRNA carriers. The lipid modification renders this otherwise ineffective low-toxic polymer a safe and effective delivery system for intracellular siRNA delivery and protein silencing. We first explore a lipid modified polymer library in adherent cells lines targeting a model protein target, the house-keeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several relevant cancer targets; P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and survivin. These initial studies in adherent cells demonstrated that although the exact formulations for efficient silencing depended on the cell line and protein target, silencing with two of the lipid-modified polymers (caprylic and linoleic acid substitutions) were consistently effective, suggesting that these carriers can be applied clinically. Fine-tuning of the siRNA/polymer composition was however critical for silencing particular targets. We then focus our efforts specifically on Acute Myeloid Leukemia (AML), where siRNA therapy development has lagged behind the cancers that are derived from attachment dependent cells, as evident in the included review of current efforts in AML siRNA therapy. We explored the feasibility of the lipid-modified carriers in AML cell lines. Efficient siRNA delivery and silencing of the model protein target, green fluorescent protein (GFP), was achieved with higher functionality than that of 25 kDa PEI, where again caprylic acid and linoleic acid substitutions stood out as the most desirable polymer substitutions. Further work demonstrated effective silencing of an AML therapeutic target CXCR4, a surface expressed adhesion protein that contributes to leukemic cell survival. The suppression of CXCR4 as well as its ligand, SDF-1 (CXCL12), resulted in a decrease in overall cell survival, which was largely attributed to a decrease in cell proliferation without enhanced effects when silencing the two targets simultaneously. The decrease in cell numbers due to CXCR4/SDF-1 silencing occurred both in the absence and presence of human bone marrow stromal cells (hBMSC), suggesting that the proposed approach would be effective in the presence of the protective bone marrow microenvironment. In more clinically related models, siRNA delivery was achieved in all human AML patient cells tested and CXCR4 silencing was demonstrated in some cases, ex vivo. The effects of silencing CXCR4 in an AML subcutaneous in vivo tumor model were also explored. Overall, we found that caprylic and linoleic lipid-substituted PEI2 can provide effective siRNA delivery to leukemic cells and can be employed in molecular therapy of leukemia targeting suitable proteins, such as CXCR4, with therapeutic outcomes. We conclude with a discussion on the further development of siRNA carriers with focus on AML therapy, describing potential enhancements that could move the field forward.

  • Subjects / Keywords
  • Graduation date
    Spring 2015
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • 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.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Specialization
    • Materials Engineering
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Heidenreich, Olaf (Molecular Haematology)
    • Hitt, Mary (Oncology)
    • Lai, Raymond (Laboratory Medicine & Pathology)
    • Uludag, Hasan (Chemical and Materials Engineering)
    • Kaur, Kamaljit (Pharmacy and Pharmaceutical Sciences)