Polymeric Nucleic Acid Delivery Systems for Reviving TRAIL Therapy for Breast Cancer

  • Author / Creator
    Thapa, Bindu
  • Targeting a single pathway is not enough to treat cancer because of compensatory mechanisms against anticancer therapy via alternative molecular pathways for survival and proliferation of malignant cells. Given the unacceptable toxicity associated with conventional therapy, nucleic acids such as plasmid DNA (pDNA), messenger RNA (mRNA) and small interfering RNA (siRNA) and their combinations could be a potential approach for specifically killing cancer cells. The idea of nucleic acid combination therapy is to simultaneously target multiple intracellular signalling pathways via overexpressing therapeutic proteins with pDNA and mRNA while silencing unwanted proteins with siRNAs. Nucleic acids, however, cannot enter cells on their own. Therefore, this thesis explored the delivery of nucleic acids and their combinations in in vitro and in vivo model using non-viral delivery systems. We first explored the galactose-based glycopolymers for the gene delivery. Optimization on size, composition (cationic vs galactose) and architectures (block vs statistical) was essential for gene delivery. Galactose containing block copolymer delivered pDNA specifically to asialoglycoprotein receptor (ASGPR) expressing hepatocytes (e.g. HepG2, Huh7.5). However, transfection efficiency of these polymers was negligible in ASGPR deficient cells. Instead, small hydrophobe and longer aliphatic lipid modified low molecular weight polyethyleneimine (PEI) were explored which efficiently delivered nucleic acid into breast cancer cells. The amount, length, and type of substitution as well as type of bond between hydrophobic group and PEI had significant impact on the transfection efficiency. Small hydrophobe propionic acid (C3) substitution on 1.2 PEI (PEI1.2-PrA) resulted higher pDNA delivery efficiency at modest substitution (0.5 to 1 PrA/PEI, mol/mol) while higher substitutions were detrimental. pDNA transfection efficiency of PEI1.2-PrA was higher than linoleic acid (C18) substituted 1.2PEI (1.2PEI-LA). However, 1.2PEI-PrA was unable to deliver siRNA whileiiialiphatic lipid linoleoyl (LA) and linolenoyl (αLA) substitution resulted higher siRNA transfection efficiencies. To target different pathways simultaneously, co-delivery of pDNA and siRNA was then explored which is more challenging due to differences in their size and structure. LA-modified thioester linked polymer (PEI1.2-tαLA) was able to co-deliver both pDNA and siRNA in in vitro and in vivo breast cancer models. Using 1.2PEI-αLA, siRNA library against 446 human apoptosis related proteins were screened and we identified, among others, two siRNAs silencing BCL2 like 12 (BCL2L12) and superoxide dismutase 1 (SOD1) which enhanced the tumor necrosis factor receptor apoptosis inducing ligand (TRAIL) induced apoptosis in breast cancer cells. Using 1.2PEItαLA, a TRAIL encoding plasmid (pTRAIL) and siRNAs targeting BCL2L12 and SOD1 were then employed as synergistic pair for breast cancer therapy. We found that co-delivery resulted higher breast cancer cell death than separate delivery without affecting normal cells. Furthermore, co-delivery of pTRAIL and BCL2L12 siRNA significantly retarded growth of breast cancer xenografts in mice. The enhanced anticancer activity was attributed to increased in situ secretion of TRAIL and sensitization of breast cancer cells against TRAIL by the co-delivered siRNAs. To mitigate the problems associated with pDNA including immunogenicity, mutagenesis due to possibility of permanent integration into genome and need for nuclear transport, all leading to low transfection efficiency, we explored mRNA delivery using PEI1.2-tαLA. Messenger RNA transfection resulted earlier and higher protein expression as compared to pDNA. Messenger RNA encoding TRAIL (mTRAIL) resulted higher cell death than pTRAIL in breast cancer cells and hBMSC modified with mTRAIL were able to kill breast cancer cells after co-culture. Overall, these studies identified polymers suitable for delivery of individual types of nucleic acids or their combination and established importance of nucleic acid combinations to support TRAIL induced apoptosis in breast cancer cells.

  • Subjects / Keywords
  • Graduation date
    Fall 2019
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
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