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.

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.

The 21st century has been called the age of genomic medicine, yet gene therapy for medicinal use remains a theory. One reason that there are no safe and effective treatments for human disease is the lack of a vehicle capable of delivering genetic material to a specific target. In nature we observe gene pathology by viral vectors, which deliver their own genetic material to specific host cells efficient at spreading the viral blueprint throughout the organism. The aim of my research into gene therapy has been to develop a synthetic vector with the delivery capability of viral vectors found in nature. This includes the ability to protect genetic cargo from modification and degradation in vivo, target to a desired cell type within a specific tissue, facilitating absorption into the cell, and delivery to the nucleus, where expression of genetic material occurs. The goal of this thesis project was to synthesize a novel vector which would selectively target the dendritic cell SIGN receptor, mirroring the method of pathogens such as HIV, which target this receptor and subsequently the immune system, resulting in chronic infection. The vector we designed contains two major components, the high mannose N-glycan Man9GlcNAc2Asn, and a peptide composed of nine amino acids: four lysine spacing residues, four lysines derivatized with acridine on the epsilon amine of their side chains, and a cysteine for conjugation to the glycan. This compound, the Man9-AcrLys Glycopeptide, was engineered to intercalate into plasmid DNA via the acridine functional groups and to bind the DC-SIGN receptor through the glycan's mannose residues. The vehicle was tested in vitro in CHO cells bearing a recombinant DC-SIGN receptor in the context of luciferase reporter gene delivery. We found that under equal treatment conditions, DC-SIGN (+) CHO cells expressed more luciferase and were 100-fold more luminescent than control DC-SIGN (-) CHO cells. My delivery method was further analyzed in a cell-sorting FACS experiment. I covalently labeled pGL3 reporter plasmid with a fluorophore, and transfected the CHO cells under typical transfection conditions. The experimental results confirmed preferential DC-SIGN mediated gene delivery.

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

No description available.

Non-covalent intermolecular interactions play a large role in determining the properties of a given system, from segmented copolymers to interactions of functionalized polymers with non-viral nucleic acids delivery vehicles. The ability to control the intermolecular interactions of a given system allow for tailoring of that system to yield a desired outcome, whether it is a copolymers mechanical properties or the colloidal stability of a pDNA-delivery vector complex. Each chemical system relies on one or more types of intermolecular interaction such as hydrogen bonding, cooperative À-À stacking, electrostatic interactions, van der waals forces, metal-ligand coordination, or hydrophobic/solvophobic effects. The following research describes the tailoring of specific intermolecular interactions aimed at altering the physical properties of segmented copolymers and non-viral gene delivery vectors. Amide containing segmented copolymers relies heavily on hydrogen bonding intermolecular interactions for physical crosslinking to impart the necessary microphase separated morphology responsible for a copolymers physical properties. Amide containing hard segments are composed of various chemical structures from crystalline aramids to amorphous alkyl amides with each structure possessing unique intermolecular interactions. Variations to either of the copolymer segments alters the copolymers physical properties allowing for tuning of a copolymers properties for a particular application. The synthetic strategies, structure-property relationships, and physical properties of amide containing segmented copolymers are thoroughly reported in the literature. Each class of segmented copolymer that contain amide hydrogen bonding groups exhibits a wide range of tunable properties desirable for many applications. The segmented copolymers discussed here include poly(ether-block-amide)s, poly(ether ester amide)s, poly(ester amide)s, poly(oxamide)s, PDMS polyamides, and polyamides containing urethane, urea, or imide groups. The structure-property relationships (SPR) of poly(oxamide) segmented copolymers is not well understood with only one report currently found in literature. The effects of oxamide spacing in the hard segment and molecular weight of the soft segments in PDMS poly(oxamide) segmented copolymers demonstrated the changes in physical properties associated with minor structural variations. The optically clear PDMS poly(oxamide) copolymers possessed good mechanical properties after bulk polymerization of ethyl oxalate terminated PDMS oligomers with alkyl diamines or varied length. FTIR spectroscopy experiments revealed an ordered hydrogen bonding carbonyl stretching band for each copolymer and as the spacing between oxamide groups increased, the temperature at which the hard segment order was disrupted decreased. The increased spacing between oxamide groups also led to a decrease in the flow temperature observed with dynamic mechanical analysis. Copolymer tensile properties decrease with increased oxamide spacing as well as the hysteresis. The structure-property investigations of PDMS poly(oxamide) segmented copolymers showed that the shortest oxamide spacing resulted in materials with optimal mechanical properties. A new class of non-chain extended segmented copolymers that contained both urea and oxamide hydrogen bonding groups in the hard segment were synthesized. PDMS poly(urea oxamide) (PDMS-UOx) copolymers displayed thermoplastic elastomer behavior with enhanced physical properties compared to PDMS polyurea (PDMS-U) controls. Synthesis of a difunctional oxamic hydrazide terminated PDMS oligomer through a two-step end capping procedure with diethyl oxalate and hydrazine proved highly efficient. Solution polymerization of the oxamic hydrazide PDMS oligomers with HMDI afforded the desired PDMS-UOx segmented copolymer, which yielded optically clear, tough elastomeric films. Dynamic mechanical analysis showed a large temperature insensitive rubbery plateau that extended up to 186 ÚC for PDMS-UOx copolymers and demonstrated increased rubbery plateau ranges of up to 120 ÚC when compared to the respective PDMS-U control. The increase in thermomechanical properties with the presence of oxamide groups in the hard segment was due to the increased hydrogen bonding, which resulted in a higher degree of microphase separation. DMA, SAXS, and AFM confirmed better phase separation of the PDMS-UOx copolymers compared to PDMS-U controls and DSC and WAXD verified the amorphous character of PDMS-UOx. Oxamide incorporation showed a profound effect on the physical properties of PDMS-UOx copolymers compared to the controls and demonstrated promise for potential commercial applications. Two novel segmented copolymers based on a poly(propylene glycol) (PPG) that contained two or three oxamide groups in the hard segment were synthesized. Synthesis of non-chain extended PPG poly(trioxamide) (PPG-TriOx) and PPG poly(urea oxamide) (PPG-UOx) segmented copolymers utilized the two-step end-capping procedure with diethyl oxalate and hydrazine then subsequent polymerization with oxalyl chloride or HMDI, respectively. The physical properties of the PPG-TriOx and PPG-UOx copolymers were compared to those of PPG poly(urea) (PPG-U) and poly(oxamide) (PPG-Ox) copolymers. FTIR studies suggested the presence of an ordered hydrogen bonded hard segment for PGG-TriOx and PPG-Ox copolymers with PPG-TriOx possessing a lower energy ordered hydrogen bonding structure. PPG-UOx copolymers exhibited a larger rubbery plateau and higher moduli compared to PPG-U copolymers and also a dramatic increase in the tensile properties with the increased hydrogen bonding. The described copolymers provided a good example of the utility of this new step-growth polymerization chemistry for producing segmented copolymers with strong hydrogen bonding capabilities. Non-viral nucleic acid delivery has become a hot field in the past 15 years due to increased safety, compared to viral vectors, and ability to synthetically alter the material properties. Altering a synthetic non-viral delivery vector allows for custom tailoring of a delivery vector for various therapeutic applications depending on the target disease. The types of non-viral delivery vectors are diverse, however the lack of understanding of the endocytic mechanisms, endosomal escape, and nucleic acid trafficking is not well understood. This lack of understanding into these complex processes limits the effective design of non-viral nucleic acid delivery vehicles to take advantage of the cellular machinery, as in the case of viral vectors. Mechanisms for cellular internalization of polymer-nucleic acid complexes are important for the future design of nucleic acid delivery vehicles. It is well known that the mammalian cell surface is covered with glycosaminoglycans (GAG) that carry a negative charge. In an effort to probe the effect of GAG charge density on the affinity of cationic poly(glcoamidoamine) (PGAA)-pDNA complexes, quartz crystal microbalance was employed to measure the mass of GAGs that associated with a polyplex monolayer. Affinity of six different GAGs that varied in the charge density were measured for polyplexes formed with poly(galactaramidopentaethylenetetramine) (G4) cationic polymers and pDNA. Results showed that the affinity of GAGs for G4 polyplexes was not completely dependent on the electrostatic interactions indicating that other factors contribute to the GAG-polyplex interactions. The results provided some insight into the interactions of polyplexes with cell surface GAGs and the role they play in cellular internalization. Two adamantane terminated polymers were investigated to study the non-covalent inclusion complexation with click cluster non-viral nucleic acid delivery vehicles for passive targeting of the click cluster-pDNA complexes (polyplex). Incorporation of adamantyl terminated poly(ethylene glycol) (Ad-PEG) and poly(2-deoxy-2-methacrylamido glucopyranose) (Ad-pMAG) polymers into the polyplex formulation revealed increased colloidal stability under physiological salt concentrations. Ad-pMAG polyplexes resulted in lower cellular uptake for HeLa cells and not two glioblastoma cell lines indicating the pMAG corona imparts some cell line specificity to the polyplexes. Ad-pMAG provided favorable biological properties when incorporated into the polyplexes as well as increased polyplex physical properties. / Ph. D.

There exists a dire need for improved therapeutics to achieve predictable and effective bone regeneration. Non-viral gene therapy is a safe method that can efficiently transfect target cells, therefore is a promising approach to overcoming the drawbacks of protein delivery of growth factors. The goal of this study was to employ cost-effective biomaterials to deliver genetic materials (DNA or RNA) in a controlled manner in order to address the high cost issues, safety concerns, and lower transfection efficiencies that exist with protein and gene therapeutic approaches. To achieve our goal, we set several aims: 1) To assess the bone regeneration capacity of polyethylenimine (PEI)-chemically modified ribonucleic acid (cmRNA) (encoding bone morphogenetic protein-2 (BMP-2)) activated matrices, compared to PEI-plasmid DNA (BMP-2)-activated matrices. 2) To explore the osteogenic potential of cmRNA-encoding BMP-9, in comparison to cmRNA-encoding BMP-2. 3) To use collagen membranes as integral components of a guided bone regeneration protocol and to enhance the bioactivity of collagen membranes by incorporating plasmid DNA (pDNA) or cmRNA encoding bone morphogenetic protein-9 (BMP-9). 4) To test whether the delivery of pDNA encoding BMP-2 (pBMP-2) and fibroblast growth factor-2 (pFGF-2) together can synergistically promote bone repair in a leporine model of diabetes mellitus, a condition that is known to be detrimental to union. 5) To investigated whether there is a synergistic effect on bone regeneration following delivery of pBMP-2 and pFGF-2, insulin and/or vitamin D. These investigations together provided new insights regarding the appropriate treatment methods for patients with fractures. Here we develop and test a non-viral gene delivery system for bone regeneration in challenging animal models utilizing a scaffold carrying PEI-nucleic acid complexes. We utilized three kinds of pDNA encoding either BMP-2, BMP-9 or FGF-2 as well as two kinds of cmRNA encoding either BMP-2 or BMP-9 formulated into PEI complexes. The fabricated nanoplexes were assessed for their size, charge, in vitro cytotoxicity, and capacity to transfect human bone marrow stromal cells (BMSCs). The in vivo functional potency of different nanoplexes embedded in scaffolds was evaluated using a calvarial bone defect model in rats, diaphyseal long bone radial defects in a diabetic rabbit model and intramuscular implantation in a diabetic rat. The results indicate that our non-viral gene delivery system induced migration and differentiation of resident cells to enhance bone regeneration. Together these findings suggest that scaffolds loaded with non-viral vectors harboring cmRNA or pDNA encoding osteogenic proteins may be a powerful tool for stimulating bone regeneration with significant potential for clinical translation.

No description available.

Including inherited genetic diseases, like lipoprotein lipase deficiency, and acquired diseases, such as cancer and HIV, gene therapy has the potential to treat or cure afflicted people by driving an affected cell to produce a therapeutic protein. Using primarily viral vectors, gene therapies are involved in a number of ongoing clinical trials and have already been approved by multiple international regulatory drug administrations for several diseases. However, viral vectors suffer from serious disadvantages including poor transduction of many cell types, immunogenicity, direct tissue toxicity and lack of targetability. Non-viral polymeric gene delivery vectors (polyplexes) provide an alternative solution but are limited by poor transfection efficiency and cytotoxicity. Microfluidic (MF) nano-precipitation is an emerging field in which researchers seek to tune the physicochemical properties of nanoparticles by controlling the flow regime during synthesis. Using this approach, several groups have demonstrated the successful production of enhanced polymeric gene delivery vectors. It has been shown that polyplexes created in the diffusive flow environment have a higher transfection efficiency and lower cytotoxicity. Other groups have demonstrated that charge-stabilizing polyplexes by sequentially adding polymers of alternating charges improves transfection efficiency and serum stability, also addressing major challenges to the clinical implementation of non-viral gene delivery vectors. To advance non-viral gene delivery towards clinical relevance, we have developed a microfluidic platform (MS) that produces conventional polyplexes with increased transfection efficiency and decreased toxicity and then extended this platform for the production of ternary polyplexes. This work involves first designing microfluidic devices using computational fluid dynamics (CFD), fabricating the devices, and validating the devices using fluorescence flow characterization and absorbance measurements of the resulting products. With an integrated separation mechanism, excess polyethylenimine (PEI) is removed from the outer regions of the stream leaving purified polyplexes that can go on to be used directly in transfections or be charge stabilized by addition of polyanions such as polyglutamic acid (PGA) for the creation of ternary polyplexes. Following the design portion of the research, the device was used to produce binary particle characterization was carried out and particle sizes, polydispersity and zeta potential of both conventional and MS polyplexes was compared. MS-produced polyplexes exhibited up to a 75% reduction in particle size compared to BM-produced polyplexes, while exhibiting little difference in zeta potential and polydispersity. A variety of standard biological assays were carried out to test the effects of the vectors on a variety of cell lines – and in this case the MS polyplexes proved to be both less toxic and have higher transfection efficiency in most cell lines. HeLa cells demonstrated the highest increase in transgene expression with a 150-fold increase when comparing to conventional bulk mixed polyplexes at the optimum formulation. A similar set of experiments were carried out with ternary polyplexes produced by the separation device. In this case it was shown that there were statistically significant increases in transfection efficiency for the MS-produced ternary polyplexes compared to BM-produced poyplexes, with a 23-fold increase in transfection activity at the optimum PEI/DNA ratio in MDAMB-231 cells. These MS-produced ternary polyplexes exhibited higher cell viability in many instances, a result that may be explained but the reduction in both free polymer and ghost particles.

Gemini surfactants are versatile gene delivery agents because of their ability to bind and compact DNA and their low cellular toxicity. The aim of my dissertation work was to develop non-invasive mucosal formulations of novel amino acid-substituted gemini surfactants with the general chemical formula C12H25(CH3)2N+-(CH2)3-N(AA)-(CH2)3-N+(CH3)2-C12H25 (AA= glycine, lysine, glycyl-lysine, lysyl-lysine). These compounds were formulated with a model plasmid DNA, encoding for interferon-γ and green fluorescent protein, in the presence of helper lipid, 1,2 dioleyl-sn-glycero-phosphatidyl-ethanolamine. Formulations were assessed in Sf 1 Ep epithelial cells. Among the novel compounds, plasmid/gemini/lipid (P/G/L) nanoparticles formulated using glycine- and glycyl-lysine substituted gemini surfactants achieved significantly higher gene expression than the parent unsubstituted compound. The key physicochemical properties, e.g. size, surface charge, DNA binding, and toxicity of P/G/L complexes were correlated with transfection efficiency. The presence of amino-acid substitution did not interfere with DNA compaction and contributed to an overall low toxicity of all P/G/L complexes, comparable to the parent gemini surfactant. A cellular uptake mechanistic study revealed that both clathrin- and caveolae-mediated uptake were major uptake routes for P/G/L nanoparticles. However, amino acid substitution in the gemini surfactant imparted high buffering capacity, pH-dependent increase in particle size, and balanced DNA binding properties. These properties may enhance endosomal escape of P/12-7NGK-12/L resulting in higher gene expression. Finally, the P/G/L complexes were incorporated into an in-situ gelling dispersion containing a thermosensitive polymer, poloxamer 407, and a permeation enhancer, diethylene glycol monoethyl ether (DEGEE). A 16% w/v poloxamer concentration produced a dispersion that gelled at body temperature and exhibited sufficient yield value to prevent formulation leakage from the vaginal cavity. The formulations were prepared with a model plasmid, encoding for red fluorescent protein, and administered topically to rabbit vagina. In agreement with our in vitro results, confocal microscopy revealed that glycyl-lysine substituted gemini surfactant exhibited higher gene expression compared to the parent unsubstituted gemini surfactant. This provided proof-of-concept for use of amino acid-substituted gemini surfactant in non-invasive mucosal (vaginal) gene delivery systems with potential therapeutic applications. These formulations will be developed with therapeutically relevant genes to assess their potential as genetic vaccines. In addition, new gemini surfactants will be developed by grafting other amino acids via glycine linkage to retain conformation flexibility and enhance endosomal escape of DNA complexes for higher transfection efficiency.