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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
No abstract available.
Nucleic acid therapies have the potential to enable the treatment of disorders previously untreatable. However, significant barriers prevent the rapid development of nucleic acid therapeutics and necessitate the use of sophisticated delivery systems. The overall objective of this dissertation is to develop more effective and tolerable lipid nanoparticles (LNPs) for nucleic acid delivery. Specifically, LNP that vary in size, stability and composition were tested for activity after subcutaneous and intravenous injection in order to optimize LNP properties. Furthermore, the incorporation of novel lipophilic pro-drugs co-delivered with nucleic acids was explored as a means to improve tolerability. The first part of this dissertation explores the use of subcutaneous administration for LNP-siRNA. There are compelling reasons to develop LNP-siRNA that can be administered subcutaneously. These include the potential for self-administration, a prolonged therapeutic window due to a depot effect and access to cell types that are in contact with the lymphatic system, in addition to tissues available through the circulation. We found that particle size and PEG steric barrier are both important for drainage from the injection site and subsequent accumulation in the liver. Although small LNP exhibited improved drainage and access to the systemic circulation, activity was impaired. The second part of this dissertation addresses this issue. The decrease in LNP activity can be attributed to the pronounced size dependent instability of LNP. By altering the amino-lipid content and the PEG-lipid used, the activity and stability of these systems can be greatly improved. The previous two parts of this dissertation identified limitations to existing LNP systems. First, administration of LNP containing nucleic acids could result in immune stimulation. Second, efforts to improve LNP activity must go beyond existing components. The last part of this dissertation proposes a general pro-drug strategy for enabling direct incorporation of additional compounds into the LNP. As an example dexamethasone, a corticosteroid commonly used to minimize infusion-related reactions, was used. Direct incorporation greatly improved the ability of dexamethasone to ameliorate immune stimulation by LNP containing nucleic acids. This work shows that when appropriately designed, LNP systems can have improved activity and tolerability, potentially expanding its clinical utility.
Lipid nanoparticles (LNP) are currently the most advanced delivery systems for enabling siRNA to be used for therapeutic applications. However, the structure of these siRNA-LNP systems has not been well defined previously. The objective of this thesis is to determine the structure of nucleic acid-LNP systems produced by a novel microfluidic mixing technique and to use this structural understanding to develop systems with improved gene silencing efficacy. The first part of the thesis focuses on determining the structure of siRNA-LNP systems produced by microfluidic mixing and the effects of varying lipid components on the structure and encapsulation properties. SiRNA-LNP were formulated using an ionizable cationic lipid, distearoylphosphatidylcholine, cholesterol and a polyethylene glycol-lipid. Cryo-TEM of siRNA-LNP produced by microfluidic mixing exhibit a solid, electron-dense core with siRNA encapsulation efficiency close to 100%. Molecular dynamics modeling indicates that the core of the particle consists of periodic aqueous compartments containing siRNA. The ability of the lipid mixture to adopt non-bilayer phases seems to be crucial for the encapsulation of siRNA. The microfluidic mixing technology was also extended to the encapsulation of plasmid DNA and mRNA. These results provide an understanding of the structure and the mechanism of formation for siRNA-LNP produced by microfluidic mixing.Since it is clear that formation of siRNA-LNP by microfluidic mixing does not require any bilayer-forming lipids, it is possible to generate particles with high cationic lipid content and bilayer-destabilizing lipids without compromising particle stability. The last part of the thesis aims to improve the gene silencing efficacy of siRNA-LNP by enhancing the endosomolytic properties of the LNP. This was attempted by two way: first by increasing the cationic lipid content of the LNP and second, by the incorporation of bilayer-destabilizing "helper" lipids in the formulation. A novel "helper" lipid dioleoyl-four amino butyric acid (DOFAB) was synthesized for this purpose. In contrary to the hypothesis, both increasing cationic lipid content and the incorporation of "helper" lipids in siRNA-LNP formulations led to decreased in vitro gene silencing activity. This leads to new questions as to role of cationic lipids and "helper" lipids play in the intracellular release of siRNA.
The androgen receptor (AR) plays a critical role in the progression of prostate cancer. This thesis is focused on investigating the ability of lipid nanoparticle (LNP) formulations of small-interfering RNA (siRNA) to silence AR (LNP AR-siRNA) in human prostate tumor cells in vitro and in LNCaP xenograft tumors following intravenous (i.v.) injection. The first part of this thesis characterized the properties of LNP AR-siRNA systems that contained the ionizable cationic lipid DLin-KC2-DMA. Inclusion of DLin-KC2-DMA was found to exhibit the most potent AR silencing effects in LNCaP cells. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to be taken up into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. Importantly, it is demonstrated that LNP AR-siRNA systems containing DLin-KC2-DMA can silence AR gene expression in distal LNCaP xenograft tumors and reduce serum prostate specific antigen (PSA) following i.v. injection at a dose level of 10 mg siRNA/kg body weight. The latter part of this thesis describes optimization of LNP AR-siRNA by stabilizing the AR-siRNA sequence through introduction of a phosphorothioate backbone and methylations of nucleotides at the 2’O position and also employing an optimized cationic lipid 3-(dimethylamino)propyl(12Z,15Z)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl]henicosa-12,15-dienoate (DMAP-BLP). In addition, specific targeting to the prostate specific membrane antigen (PSMA) on LNCaP cells was made possible via chemical conjugation of a small molecule 2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) to the PEG-lipid formulated into LNP AR-siRNA. With the incorporation of the DUPA-targeting moiety, a 5-fold increase in cellular uptake was observed in LNCaP cells in vitro, as well as a dramatic improvement in AR knockdown. The PEG-lipid employed in formulating the LNP was also optimized to produce longer circulation lifetimes that result in improved accumulation at the distal tumor site. It is shown that as a result of these improvements the doses of siRNA employed in LNP-siRNA systems could be reduced by a factor of two as compared to previous systems. In addition improved reductions in serum PSA, cellular proliferation, and AR levels were also observed. These results support the potential clinical utility of LNP-siRNA systems to silence the AR for treatment of advanced prostate cancer.
Master's Student Supervision (2010 - 2018)
No abstract available.