Lipopolymer Facilitated RNAi Therapy for Myeloid Leukemia
Creators: Ansari, Aysha S
Publisher: name: University of Alberta Library
Year: 2023

<p>RNA interference (RNAi) technology is gaining momentum as a revolutionary therapeutic tool in the clinic with five siRNA-based drugs attaining approval in the past five years. In contrast to exploiting proteins as targets like most conventional treatments, RNAi therapeutics exert their actions towards disease-driving aberrant genetic fingerprints. While specific small molecule inhibitors represent a great stride in improving survival for myeloid leukemia patients, they are rendered futile in the face of evolving landscape of acquired mutations leading to drug resistance, necessitating the pursuit of alternative approaches. Implementing RNAi therapy requires a robust siRNA carrier to evade physiological barriers to reach site of action and mediate cellular entry, besides a potent siRNA sequence. This thesis first provides a review of the physical methods and biomaterials that have been developed for anchorage-independent cells that are more resilient to nucleic acid transfection than anchorage-dependent cells, followed by a summary and perspective on exploiting oncogenic fusion genes as RNAi targets in myeloid leukemia. Lipid-grafted on low molecular weight polyethyleneimine (PEI) polymers (lipopolymers) were then investigated as siRNA delivery vehicles targeting recurring fusion genes in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). The physicochemical characteristics of water-insoluble cholesterol-grafted 1.2 kDa PEI (PEI-Chol) and their therapeutic efficacy in targeting the BCR-ABL oncogene was explored in CML. PEI-Chol/siRNA complexes were significantly smaller in size and possessed higher cationic charge compared to the parent polymer. The siRNA binding capacity and complex dissociation sensitivity of PEI-Chol shared a direct and inverse relationship, respectively, with increasing cholesterol content of the lipopolymers. The therapeutic efficacy of PEI-Chol/siRNA complexes was reflected in their ability to effectively silence the reporter green fluorescent protein (GFP) gene and BCR-ABL as well as significantly inhibit colony formation post BCR-ABL silencing. Efforts were then focused specifically on FLT3-ITD driven AML, which is present in about a quarter of newly diagnosed AML patients and is associated with a high relapse rate and poor clinical outcomes. Screening of a large library of lipopolymers revealed linoleic acid (LA) and α-linolenic acid (αLA) to be the most suitable substituents for siRNA therapy in FLT3-ITD AML cells. The identified lipopolymers demonstrated delivery of siRNA targeting FLT3 and significant suppression of FLT3 gene and protein levels post treatment with lipopolymer/siRNA complexes, leading to a prominent decline in cell proliferation and induction of apoptosis in cell lines. Similar FLT3 suppression in mononuclear cells derived from FLT3-ITD positive AML patients showed reduction in colony forming ability, suggesting not only effective delivery to primary cells but also functional activity in the leukemic progenitor subpopulation. The absence of any discernible physiological response in wild-type FLT3 AML cells and mononuclear cells isolated from the peripheral blood of healthy individuals bore evidence of the sensitivity and specificity of lipopolymer/siFLT3 complexes. Combining lipopolymer/siFLT3 complexes with the chemotherapeutic drug daunorubucin, and FLT3-specific small molecule inhibitors midostaurin and gilteritinib exhibited significant improvement in therapeutic response while permitting the use of a lower dose of the clinical agents. Furthermore, lipopolymer mediated siFLT3 delivery in a subcutaneous FLT3-ITD AML xenograft model led to a prominent decline in tumor volumes as a monotherapy, and enhanced median survival in combination with gilteritinib, providing a proof-of-concept for clinical translation. Repressing aberrant fusion oncoprotein activity through restricting expression levels by deploying lipopolymer/siRNA complexes is, therefore, a therapeutically viable option and could be an effective choice in treating myeloid leukemias. The thesis is concluded with a discussion and future research directions that will aid in enhancing performance and advancing the lipopolymer facilitated RNAi therapeutic platform into the clinic.</p>