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Lipid based non-viral delivery systems are potentially of great importance to the development of an effective and versatile therapeutic gene treatment. Many difficulties are faced in designing efficient lipid based gene delivery systems and addressing the problem of endosomal escape is one key area where efficiency could be greatly improved Molecules aimed at inducing aggregate disruption in response to changes in pH, or metal ion concentration, have been synthesised. Series of compounds were prepared based on cationic 5-alkyl-2-methylaminoalkyl pyridine amphiphiles and amphiphilic C(_8)-C(_16) EDTA dialkylamides. The critical aggregation concentration of each species was measured and a series of experiments undertaken, designed to assess the extent of pH, or metal ion concentration, induced aggregate disruption effects. The experiments were carried out by including the molecules to be examined as small mole percent co-aggregates in micellar and liposome model systems. Each group of compounds appears to exhibit disruption effects on the more strongly ordered bilayer membrane systems, with the EDTA based compounds displaying the most consistent pH dependent disruption. However, the more dynamic micellar aggregate models were less sensitive to disruption using the particular observation method employed.

The ability to deliver DNA to target cells creating therapeutic effects remains an important goal in the field of gene therapy. A majority of clinical trials to overcome this issue have utilized viral vectors due to their efficiency at DNA delivery and ability to create high levels of gene expression. However, their inherent toxicity and a several clinical trials leading to patients contracting new diseases from the treatment have greatly hindered the progress of viral gene therapy. Non-viral gene delivery agents have a much better safety profile, but are also much less efficient at delivering DNA, leading to low gene expression. The reason for this low expression is the numerous barriers that must be overcome to achieve gene expression: circulation, tissue specific accumulation, internalization, release of DNA cargo, and nuclear localization. While peptides are currently being improved upon, enhancing binding and the ability to protect DNA, they are still deficient when it comes to tissue specificity. Numerous targeting methods, including the use of lectins, antibodies, aptamers, and peptides, have been designed to deliver molecules to a specific research. Research to incorporate targeting ligands onto non-viral gene delivery vectors is abundant in the literature; however, successful site specific gene delivery has not been achieved. The somatostatin receptor 2 (SSTR2) ligand, octreotide, is a well-researched eight amino acid peptide that has extensive SAR data available. Also, the receptors have been well characterized and octreotide is used clinically in the radioscintigraphy imaging of brain tumors. While well researched, there are unexplored opportunities to utilize octreotide to enhance non-viral gene delivery vectors. The overall scope of this thesis is to develop and synthesize non-viral gene delivery peptides conjugated to octreotide creating receptor mediated targeting of DNA polyplexes to create tissue specific accumulation. Initial experiments indicated that attachment of octreotide to the polycationic peptide WK18 does not inhibit affinity for the SSTR2 receptor. Therefore, peptides were designed and synthesized to attach octreotide onto polyacridine peptide (Acr-Lys)6. Polyplex characteristics were unchanged by the incorporation of octreotide, and exhibited very low genotoxic effects compared to the in vitro gene delivery agent PEI. Competitive binding assays suggested a stoichiometric, ligand, and temperature dependent accumulation of polyplex on SSTR2 expressing cells, but gene expression could not be achieved. The success of (Acr-Lys)6octreotide, led to the synthesis of a di-maleimide-PEG attached to each end by (Acr-Lys4)3Acr-Lys-Cys or Cys-Gly5octreotide in attempts to create distance, and better ligand availability for the receptor, by expressing octreotide away from the polyplex. Testing of this peptide in PEGylated polyplex ad-mixtures verified that separating the DNA binding peptide from octreotide did lead to better inhibition of binding to DAOY cells in a competitive binding study. However, transfection assays with this compound showed background levels of gene expression. Although gene expression was not achieved, the synthetic strategy to create a molecule incorporating a DNA binding peptide, ligand, and PEG to create better ligand presentation to its receptor when incorporated into PEGylated polyplexes is an important step in the design of gene delivery vectors.

These investigations are focused on the design and formulation of novel nonviral lipopolyamine vectors capable of efficiently and safely delivering DNA to the nucleus, and siRNA to the cytoplasm, in two tissue cultured (primary and cancer) cell lines. The thesis starts with a focussed literature review on the non-viral gene therapy (NVGT) vectors currently used in the formulation of DNA and siRNA. The first experimental part tests the ability of our novel lipospermines in NVGT, this includes structure-activity relationship (SAR) studies changing the: position, length, saturation or symmetry of the fatty chains of N4,N9-diacyl, N1,N12-diacyl and N4,N9-dialkyl spermines. The ability of these lipospermines in DNA condensation is investigated using ethidium bromide fluorescence-quenching, and gel electrophoresis (including gel shift and DNase protection) assays followed by nanoparticle characterization techniques (particle size and zeta potential). Transfection efficiency of pEGFP (using FACS) and cytotoxicity (using MTT) were studied in both cancer and primary cell lines and compared with Lipogen™ (N4,N9-dioleoyl spermine). Some of these novel lipospermines are shown to be as good as, but not better than N4,N9-dioleoyl spermine as efficient DNA transfecting agents. N4,N9-Dioleoyl spermine is the best transfecting agent from the all tested novel lipospermines displaying the lowest N/P ratio, highest transfection efficiency and the lowest cytotoxicity on both tested cell lines. We extended this SAR study to examine the same lipospermines in siRNA delivery. The ability of these compounds to bind siRNA was studied using the RiboGreen intercalation assay followed by similar nanoparticle characterization techniques. Transfection efficiency for delivery of Label IT® RNAi Delivery Control (using FACS) and cytotoxicity (MTT) were also studied in both cancer and primary cell lines, and compared with a market leader siRNA transfecting agent Trans-IT™. Twelve of these non-viral vectors, led by N4,N9-dieicosenoyl spermine and N4,N9-dierucoyl spermine, showed both transfection efficiency and cell viability over 75%.

It is well known that multivalent cations can cause DNA to condense from solution to form high-density nanometer scale particles. However, several fundamental questions concerning the phenomenon of DNA condensation remain unanswered. DNA condensation in vitro has been of interest for many years as a model of naturally occurring DNA packaging (e.g. chromatin, sperm head and virus capsid packing). More recently, DNA condensation has been of interest in optimizing artificial gene delivery, where packaging genes to an optimal size is essential to developing efficient uptake and delivery systems. The research presented in this dissertation provides an in depth biophysical study of the factors that control DNA condensate size and morphology. Millimolar changes in the ionic strength of the solution were found to alter the size of toroidal condensates. Variations in the order of addition of the counterions also significantly changed the size and morphology of the condensates. Studies were also performed to investigate the effects of static curvature and increased DNA flexibility on DNA condensation. These include the addition of static bending by sequence directed curvature, dynamic bending through protein-DNA interactions and reducing DNA persistence length by condensing single-stranded DNA. Several new models of DNA condensation are proposed based on the experimental data presented in this thesis.

The effectiveness of most chemotherapeutic regimens is limited by the toxicity of the therapy to normal healthy cells. Therapies to selectively modulate abnormal T cells bearing the interleukin 2 receptor (IL-2R) have been developed to treat diseases associated with aberrant immune response. This study describes the development and optimization of a targeted gene or oligonucleotide therapy vehicle to IL-2R bearing T cells for selective elimination of these cells. In this work, a monoclonal antibody to the IL-2R was used to target the oligonucleotide delivery vehicle which consisted of a polyamidoamine dendrimer. Optimization of the delivery vehicle involves understanding the factors that govern its association with oligonucleotide, the pathway of IL-2R endocytic trafficking, and the stability of the oligonucleotide in the biological milieu. Oligonucleotide stability in a cellular environment was examined intra- and extracellularly. Results showed that the rate of intracellular degradation of oligonucleotides was much greater than extracellular degradation. Binding of oligonucleotides to dendrimers was demonstrated as a function of dendrimer generation. The total binding capacities for dendrimers differed depending upon dendrimer size and surface group, whereas equilibrium binding affinity was comparable for all dendrimers tested. Binding of oligonucleotide delivery vehicle to the cell surface and subsequent internalization was inversely related to dendrimer size, and in all cases, significantly less than binding and internalization of the natural ligand for the IL-2R. Based on experimental results, a kinetic model of the delivery vehicle was derived which includedthe dependence of binding and internalization on dendrimer size and surface charge and intracellular degradation of oligonucleotide. Based on model predictions, we show that larger dendrimers carry more oligonucleotide than the smaller dendrimer vehicles, and delivery is more effective with larger vehicles. This work establishes our ability to predict the effects of different delivery vehicle properties on oligonucleotide delivery and aids in the development of design criteria for new vehicles for delivery of antisense, siRNA, or genes to IL-2R bearing cells.

abstract: Gene delivery is a broadly applicable tool that has applications in gene therapy, production of therapeutic proteins, and as a study tool to understand biological pathways. However, for successful gene delivery, the gene and its carrier must bypass or traverse a number of formidable obstacles before successfully entering the cell’s nucleus where the host cell’s machinery can be utilized to express a protein encoded by the gene of interest. The vast majority of work in the gene delivery field focuses on overcoming these barriers by creative synthesis of nanoparticle delivery vehicles or conjugation of targeting moieties to the nucleic acid or delivery vehicle, but little work focuses on modifying the target cell’s behavior to make it more amenable to transfection. In this work, a number of kinase enzymes have been identified by inhibition to be targets for enhancing polymer-mediated transgene expression (chapter 2), including the lead target which appears to affect intracellular trafficking of delivered nucleic acid cargo. The subsequent sections (chapters 3 and 4) of this work focus on targeting epigenetic modifying enzymes to enhance polymer-mediated transgene expression, and a number of candidate enzymes have been identified. Some mechanistic evaluation of these targets have been carried out and discussion of ongoing experiments and future directions to better understand the mechanistic descriptions behind the phenomena are discussed. The overall goal is to enhance non-viral (polymer-mediated) transgene expression by modulating cellular behavior for general gene delivery applications. / Dissertation/Thesis / Doctoral Dissertation Chemical Engineering 2016

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