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UBC Theses and Dissertations

Optimization of the intracellular delivery properties of non-viral DNA carrier systems Sandhu, Ammen Preat

Abstract

A central problem faced by the gene therapy field is the lack of efficient non-toxic methods of delivery of DNA-based macromolecules, such as antisense oligonucleotides and plasmid DNA in vivo. A major reason for this is that currently available non-viral delivery systems are relatively inefficient agents for intracellular delivery of associated DNA. This thesis examines methods to improve the intracellular delivery properties of lipid-based delivery systems for DNA-based macromolecules. In Chapter 2, the efficiency of stabilized antisense-lipid particle (SALP) delivered antisense oligonucleotide (ASODN)-mediated downregulation of a target gene, PKC-a, was evaluated in vitro and in an in vivo mouse model. Results obtained confirmed that significant levels of SALP encapsulated ASODN are taken up by cells; however, a majority of the oligonucleotide remains locked in endosomal/lysosomal compartments. Poor intracellular delivery by SALP systems was also supported by the lack of PKC-a mRNA downregulation following in vitro treatments with SALP-delivered ASODN targeting PKC-a. The relative efficiency of SALP delivery of ASODN in the in vivo liver model was not consistent with the results observed in vitro, since there was evidence of a decrease in PKC-a mRNA levels in the liver tissue following SALP delivery. However, the levels of antisense activity observed in the in vivo model were complicated by non-sequence-specific effects. In Chapter 3, focus was shifted towards studying the intracellular delivery properties of stabilized plasmid-lipid particles (SPLP) since assays to quantify delivery of a plasmid encoding for the luciferase reporter gene are more unambiguous, sensitive and relatively straightforward as compared to methods to determine ASODN activity. The intracellular release parameter (IRP) was employed to examine how changes in the lipid composition and transfection parameters of SPLP affect intracellular delivery. It is shown that the major factor limiting the intracellular delivery properties of the standard SPLP system is the presence of the non-exchangeable PEG-CerC2 o molecules. Another major finding is that the addition of 8 mM C a 2 + into the transfection medium increased the IRP parameter of SPLP by 100-500 fold regardless of the SPLP lipid composition. In Chapter 4, the significant enhancement in intracellular delivery of SPLP in the presence of C a 2 + was studied further to determine the underlying mechanisms behind its activity. It is shown that the C a 2 + effect could be modulated by changes in the serum content in the cell culture medium but is not related to modifications in cell uptake levels of SPLP. The C a 2 + effect is attributed to the formation of calcium phosphate precipitates during the transfection procedure; however the effect does not involve calcium phosphate mediated destabilization of SPLP. It is also demonstrated that calcium phosphate precipitates enhance the intracellular delivery of neutral macromolecules by facilitating their release from endosomal/lysosomal compartments. In Chapter 5, the strategy to enhance the intracellular delivery properties of SPLP using pH-sensitive polymers was explored. It is demonstrated that coadministration of poly(ethylacrylic acid) (PEAA) polymers with SPLP increases gene expression by up to 1000-fold in BHK cells. Further, an acylated derivative of the PEAA polymer was successfully post-inserted into the SPLP system at efficiencies of 70-80% without significantly affecting SPLP particle size or DNA protective qualities. Transfections with SPLP containing post-inserted acylated PEAA increased gene expression by up to 80-fold, which was not related to changes in cell uptake levels.

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