Adeno-associated virus (AAV) is a 25 nm replication deficient DNA virus most commonly studied for human gene therapy applications. The work detailed in this thesis investigates the controlled delivery of AAV from surfaces for both transduction and biosensing applications. First, AAV was tested for compatibility with substrate-mediated gene delivery approaches. Two reverse transduction applications were investigated: (1) spatial localization of cells and virus vectors for tissue engineering applications and (2) live cell genetic microarrays. To drive the proper differentiation and assembly of cells within tissue engineering constructs, gene expression patterns may need to be tightly regulated. To localize adhesive proteins and AAV, polydimethylsiloxane stamps and protein adhesive alkanethiols were used. By adsorbing AAV onto adhesive proteins, including human fibronectin, laminin, collagen I, elastin and poly-l-lysine, both cell adhesion and gene delivery were localized to a defined pattern. Gene delivery was efficient on all protein surfaces, with higher expression observed on laminin surfaces. AAV was also patterned using a robotic spotter to create live cell genetic microarrays, creating localized cell islands expressing GFP. This potentially high-throughput technique could be extended to study complex genetic interactions within cells, such as stem cells or induced pluripotent stem cells. Additionally, AAV was explored as a biosensor by modifying virus output functionalities. Wild-type AAV2 externalizes an N-terminus region containing a phospholipase A2 (PLA2) domain during intracellular processing, allowing the virus to escape the endosomal pathway and deliver genetic cargo. This externalization can be replicated outside of cells through heat treatment. AAV2-ΔPLA2-His was created by replacing the PLA2 domain with a nickel binding hexahistidine tag. This replacement allows heat-treated mutant virus to bind a nickel affinity column. Finally, directed evolution was used to (1) improve the ability of AAV to deliver genes into target cells or (2) alter AAV biosensor inputs. Virus libraries were created using error-prone polymerase chain reaction (EP-PCR) to introduce random amino acid modifications into the protein capsid. The error rate for these libraries was estimated to be between 5-7 errors per cap gene. Combining surface immobilization with directed evolution could allow for precise control of AAV for gene delivery and biosensing.

<p>Three-dimensional (3D) tumor models aim to reduce the need for animal models for drug and gene delivery studies. However, many models are not conducive to environmental manipulation and may not be easily adapted for <italic>in situ</italic> microscopic analysis of transport phenomena. One goal of this study was to develop a 3D tumor model that can mimic 3D cell-cell interactions to mimic native tumor tissues. </p><p>To this end, a novel 3D microfluidics-based tumor model was created which allowed the overnight culture of a high density of tumor cells and could be used for small molecule penetration studies. This microfluidic device facilitated the loading of B16.F10 tumor cells in a densely-packed three-dimensional arrangement in a micro-channel which was accessible for nutrient supply via channels on either side through which culture media was continuously infused. Cell volume fraction in the micro-channel was determined via nuclear staining and counting of cells immediately after loading and after a 12-hr culture period. The average volume fraction of cells in this model was 0.32 immediately after loading and 0.26 after 12-hr culture. The values are comparable to cell volume fractions of the <italic>in vivo</italic> B16.F10 tumor previously measured in our lab. The reduction in cell volume fraction after overnight culture was due to the change in cell morphology to become more elongated after time in culture. Cell-cell adhesions appeared to have formed during culture, resulting in more uniform packing. </p><p>Sodium fluorescein dye was used as a drug analog and the extent of penetration of this fluorescent molecule through the cell compartment was assessed through microscopy. The dye was introduced on one side of the cell micro-channel and fluorescence images were captured for generation of concentration profiles in the cell compartment. Results showed that dye penetration through the cell chamber was greatly limited by the presence of the 3D cell culture and a linear concentration profile was achieved across the cell compartment. Also, the concentration of sodium fluorescein in the cell compartment of the 12-hr microfluidic cell culture was appreciably lower than the concentration in the cell compartment when the dye was introduced immediately after loading cells. These results suggest that the proposed tumor model shows significant resistance to dye penetration and could prove to be extremely useful for mimicking tumor tissue resistance to drug penetration via diffusion.</p><p>There are many barriers to gene delivery to tumors which highlight the importance of selecting an effective gene carrier system. Some pathogenic bacteria have been investigated as gene delivery vectors because of their innate ability to selectively proliferate in tumor environments. However, pathogenicity concerns arise when trying to achieve therapeutic levels of gene expression. It has been shown that non-pathogenic bacteria such as <italic>E. coli</italic> can be engineered to invade mammalian cells and participate as gene delivery vehicles. Hence, the second part of this research project involved the use of the newly developed microfluidic 3D tumor model previously described to visualize the transport behavior of invasive (inv+) and non-invasive (inv-) <italic>E. coli</italic>. The inv+ bacteria harbored a plasmid containing the inv gene encoding the protein invasin that binds to &beta₁ integrin receptors on the surface of mammalian cells resulting in the phagocytosis of invasin-expressing bacteria by normally non-phaogcytotic cells. Two tumor cells lines were used: B16.F10 and EMT6, which have been shown to differ in expression of &beta₁ integrins. The bacteria were also engineered to express mCherry for fluorescent detection.</p><p>A suspension of tumor cells and bacteria was loaded into the microfluidic device and cultured for 12 hrs before imaging bacteria distribution throughout the cell culture. Proliferation of inv+ bacteria was generally uniform throughout the cell compartment in the B16.F10 model and bacterial cells were primarily concentrated outside of cells. Bacteria that were internalized did not appear to migrate far from the plasma membrane of the tumor cell. The non-invasive bacteria proliferated to a much greater extent than the invasive form and this proliferation was also generally uniform throughout the cell compartment. Proliferation of both invasive and non-invasive bacteria in the EMT6 model was less uniform than in the B16.F10 model. Overall bacterial concentration appeared to be lower in the EMT6 model. Viability staining after bacterial infection showed that tumor cells in the 3D model were able to maintain viability despite bacterial cell proliferation. </p><p>An additional assay was conducted in culture plate wells to determine the effect of chemical factors secreted by tumor cells on bacterial cell proliferation. The results of this assay revealed that tumor cells may be secreting anti-microbial factors that inhibit the proliferation of bacteria and that the binding of invasin-expressing <italic>E. coli</italic> to tumor cells may further promote the release of these factors.</p><p>The results of this study suggest that tumor cell type plays a major role in the distribution and proliferation of bacteria in a 3D environment. The ability to visualize bacterial spread throughout a 3D tumor model will prove to be useful for observing the effect of various genetic modifications on the transport and gene delivery efficiency of <italic>E. coli</italic>.</p> / Dissertation

This study was conducted to determine if electroporation could deliver membrane-impermeant molecules intracellularly to intact, physiologically competent monolayers that mimic the intestinal epithelium. The long-term effects of electroporation on these monolayers were studied to determine the kinetics with which monolayers recover barrier function. The ability of electroporation to introduce biologically active molecules, e.g., plasmid DNA and siRNA, into these monolayers, to either express a protein of interest or modify cellular function, was also studied. Results showed that intracellular uptake of calcein, a small tracer molecule, and bovine serum albumin, a globular protein, occurred uniformly throughout the monolayers and increased as a function of voltage, pulse length, and pulse number. There was no significant difference in uptake resulting from single and multiple pulses of the same total exposure time. Barrier function recovery depended on the electroporation conditions applied, with some monolayers recovering normal physiologic function within a day. Electroporation also increased the permeability of the monolayers to calcein and BSA, possibly through a combination of increased paracellular and transmonolayer transport. When compared to cationic lipid transfection (lipofection), transfection of intestinal epithelial monolayers with reporter plasmids by electroporation was more efficient in situations where high concentrations of DNA, and as a result, higher levels of expression were needed. Although uptake of DNA was high after electroporation and increased with increasing amounts of DNA, overall expreseion efficiency was still low (~3%). Electroporation-mediated transfection of intestinal epithelial monolayers with a plasmid that expressed inflammation inhibitor protein, IκВα was not always successful, probably because of low levels of protein expression. Introduction of the much smaller siRNA molecules into the monolayers by electroporation, on the other hand, was very successful at inhibiting the production of the nuclear envelope proteins lamin A and lamin C. The results of these experiments demonstrated that electroporation can introduce a wide variety of molecules intracellularly into model intestinal epithelia. These results should be useful to identify optimal electroporation conditions for transporting drugs, proteins, and genes into intestinal and, possibly, other epithelia for local drug and gene therapy, as well as for development of improved models of intestinal epithelium.

Research in the field of non-viral gene delivery has demonstrated that a deeper understanding of the fundamental processes of nanoparticle assembly is required in order to improve their efficacy. While gemini nanoparticles (gemini NPs) and other non-viral delivery systems have been vigorously characterized using several techniques, our knowledge is still incomplete. The first objective of this study was the development of new methodology using fluorescence correlation spectroscopy (FCS) to investigate the stages of gemini NPs assembly. It was demonstrated that by labeling the plasmid, different stages of gemini NP assembly from the gemini-plasmid pre-complex (GP) to the final gemini nanoparticle (or gemini-plasmid-lipid complex; GPL), could be studied. Based on diffusion coefficients and particle numbers extrapolated from the autocorrelation function (ACF), FCS was able to determine that each phase of assembly had distinct characteristics. The FCS study using 12-3-12 gemini surfactant showed that both the diffusion coefficient and particle number of GPs (0.98??0.31 x 10-12 m2/s) was significantly lower than the final GPL (3.11??0.41 x 10-12 m2/s). Based on the Stokes-Einstein equation the particle size was calculated to be 300-500 nm for GP and 200-300 nm for GPLs. The raw intensity histograms showed that both GPs and GPLs are composed of multiple plasmids. Furthermore the study showed that the final GPLs contain fewer plasmids compared to the intermediate GP. FCS results were validated by using existing characterization methods including dynamic light scattering (DLS), zeta potential and dye exclusion assays. The second objective involved the detailed characterization of gemini NP. Nine different gemini surfactants and two different phospholipids were used in a systematic study to assess the effect of gemini surfactant and lipid structure on the final morphology of gemini NP. The study revealed that gemini surfactant structure had a strong effect on structure of GP intermediates, but addition of phospholipids resulted in the formation of uniform gemini NPs. Based on the results of this study a new model for GP and GPL assembly is proposed based on the formation of supramolecular aggregates of gemini-plasmids, governed by gemini surfactant chemical structure, and dispersed by phospholipids to form GPLs.

The feasibility of most gene therapy strategies depends on the efficient delivery of DNA to target cells and tissues. Current gene delivery carriers can be divided into two classes: viral and non-viral delivery systems. Although the viral carriers are highly efficient due to their invasive nature, safety concerns may restrict their application in clinical settings. Synthetic non-viral carriers attract increasing attention because they are less toxic and allow readily modification. Non-viral carrier mediated gene delivery involves several processes. They must condense DNA into small particles, allow membrane penetration and protect DNA from extracellular and intracellular degradative enzymes. In the present study, a small library of carriers containing various combinations of cell penetrating peptide TAT, SV40 large T protein nuclear localisation signal (NLS) and cationic dendrimer of 7 lysine residues (DEN) was synthesised and tested for their ability to deliver DNA to mammalian cells. We evaluated the contribution of each component as well as the combination of the components on DNA condensation, uptake and gene expression. It was found that all carriers condensed DNA and protected DNA from DNase degradation. We showed that the TAT peptide was essential, but not sufficient, for uptake of exogenous DNA. The addition of either NLS or DEN significantly enhanced uptake. The most efficient carrier contained all three components (DEN-NLS-TAT). The carriers were able to deliver DNA in the presence of serum and were non-toxic to cells at up to 30 μM. However, for those peptides that facilitated DNA uptake, the complexes were targeted to intracellular compartments that required a fusogenic agent, such as chloroquine, before gene expression was observed. Modifications were introduced to the initial carrier library in order to circumvent the chloroquine dependence. The addition of cell penetrating peptide penetratin, virus derived fusogenic peptide or lipoamino acid C12 enhanced either DNA uptake or endosomal release. However, none of the modified carriers were able to produce high level transgene expression in the absence of chloroquine. We also found that the carriers containing lipid components were able to deliver DNA to T-lymphocytes derived cells, which are usually resistant to transfection. However, the toxicity of the lipid-based carriers needs to be reduced before further application. We also evaluated the function of chloroquine as a gene expression enhancer. We demonstrated that chloroquine did not enhance expression solely by promoting endosomal release. This was supported by the fact that fusogenic peptide and endosomal disruptive reagents (bafilomycin A1 and monensin) did not improve gene expression. Other properties of chloroquine, such as DNA protection and transcription enhancement, may also contribute to gene expression. We characterised the uptake mechanism of DEN-NLS-TAT in HeLa cell lines. We found that the uptake of DEN-NLS-TAT/DNA complex in HeLa cell line was mainly via receptor-mediated endocytosis and caveolae endocytosis. Moreover, various intracellular processes, such as intact cytoskeleton and microtubule network, tyrosine and PI 3 kinase activity, and membrane cholesterol were also required for the uptake of the carrier/DNA complex. In conclusion, the results from the present study demonstrated that multi-component peptide-based carriers are versatile carriers for the delivery of plasmid DNA in human cells. The results have improved our understanding of the role of chloroquine as a widely used gene expression enhancer which may be useful in the future improvement of non-viral gene delivery carriers. A strategy to overcome the dependence on chloroquine for gene expression or reduce the toxicity of chloroquine will be necessary for further in vivo applications. The current carrier library may also be used to delivery other cargos such as siRNA or protein to human cells.

When an electric field is locally applied to tissues in vivo the uptake of exogenous DNA can be greatly increased. This approach to gene transfer, called electroporation (EP) or gene electro transfer (GET), has potential applications in the treatment of skin disorders, vaccinations, some types of cancer and metabolic diseases. The eect of electric elds on cells and tissues has been studied and related to the uptake of DNA. Tissue impedance changes have been measured as a result of electroporation. The aim of this study is to explore the predictive accuracy of impedance spectroscopy for the success of GET. Mice were used in this study for their histological similarities to human skin. The mice were injected with plasmid DNA coding for luciferase and given one of a series of electroporation treatments varying the number and intensity of the electric pulses delivered. The number of pulses delivered was based upon impedance measurements taken during the EP procedure. Mice were then imaged to quantitatively measure the luminescence resulting from the gene delivery procedure at intervals of 2, 4, 7, 10 and 14 days after treatment to quantitatively determine the biological response to gene electrotransfer. Increased luminescence was noticed in treatment groups compared to injection only groups, and the most eective treatments resulted from a feedback mechanism based upon percentage changes in skin impedance. The relationship between impedance change and gene expression suggested that treatment can be improved based on impedance measurements taken during the EP treatment.

Advances in molecular medicine have produced a large amount of information about genes that translate to therapeutic molecules when expressed in living cells. There is an increasing interest in nonviral methods for gene delivery, to address all concerns on non-toxic, easy, and possibly efficient delivery systems. In this paper we introduced a new attractive approach for non-viral transferring of genetic materials on demand. By using lipofectin reagent (1:1 molar ratio of DOPE:DOTMA. DOPE: L-α-doleoyl posphatidylethanolamine; DOTMA: N-[1-(2,3-dideyloxy) propyl]-n,n,n-trimethylammonium chloride), the lipid/DNA complexes (lipoplexes) can be electrostatically adsorbed on the gold microelectrode surface. The resulting lipoplexes molecules can be subsequently removed from the surface by applying -1.0 V (vs. Ag/AgCl) in physiological phosphate buffer medium (pH 7.4). This electrochemically controlled-release process has been extensively examined by gel electrophoresis (GE), electrochemical quartz crystal microbalance (EQCM), infrared spectroscopy (IR), and square wave voltammetry (SWV) techniques. The lipoplex composition has been addressed for efficient gene delivery protocol, based on their different charge ratios. The results from different techniques coincided, as also verified by the repetitive control experiments. This in-vitro electrically - triggered release protocol for genetic material offers the current gene delivery arsenal a new, simple, and non-viral alternative.

This study demonstrates the direct delivery of plasmid gene constructs into spinal motor neurons utilizing retrograde axoplasmic transport. The plasmid vectors contained the Lac Z gene under the control of both the Rous sarcoma virus (RSV) and Simian virus (SV)40 promoters. β-Galactosidase expression was observed in α and γ motor neurons by histochemical staining following direct injection into the sciatic nerve or gastrocnemius muscle. The presence of LacZ gene constructs was confirmed by the polymerase chain reaction (PCR). The ability to introduce gene constructs into motor neurons allows for the study of gene regulation and permits the development of gene therapy strategies for motor neuron diseases including the spinal muscular atrophies (SMA) and amyotrophic lateral sclerosis (ALS).

No description available.

Wide literature precedence exists for polymers containing electrostatic interactions and polymers containing hydrogen bonding motifs, however the combination of electrostatic and hydrogen bonding interactions is not widely investigated in current literature. Polyelectrolytes containing hydrogen bonding groups are expected to exhibit properties of both classes of supramolecular interactions. A series of adenine- and thyminecontaining PDMAEMA and tert-butyl acrylate copolymers were synthesized to investigate the effect of incorporating hydrogen bonding groups into a polyelectrolyte. Incorporation of the styrenic nucleobases significantly affected the solubility of these copolymers on aqueous solutions and showed salt-triggerability with higher contents of these groups. Polyelectrolytes are capable of binding and condensing DNA through electrostatic interactions with the negatively charged phosphate groups of the DNA backbone; however a high degree of cytotoxicity is also often observed for these gene delivery systems. The high level of cytotoxicity is attributed to high degree of cationic character for the polyplexes formed with these systems according to the proton-sponge hypothesis. One method of reducing the overall cationic character for these systems is incorporation of non-electrostatic binding mechanisms such as hydrogen bonding. A series of nucleobase-containing PDMAEMA copolymers were utilized in order to investigate the effect of incorporation of these groups on the cell viability, binding efficiency, and transfection efficiency of PDMAEMA. / Master of Science