Browsing by Subject "Gene Delivery"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Charged Glycopolymer Materials for Epidermolysis Bullosa
    (2017-12) Boyle, William
    Improved delivery of therapeutic nucleic acid payloads to cells could lead to dramatically improved clinical outcomes for patients suffering from genetic disorders. This work focuses on the use of a trehalose-containing cationic glycopolymer, termed Tr4, to transfect clinically relevant cell types. In particular, the development of gene delivery methods to improve the transfection of cell types associated with the skin disease epidermolysis bullosa are investigated. The sulfated glycosaminoglycan, heparin, is show to form ternary complexes with pDNA and Tr4 leading to dramatically increased transfection efficiency in primary fibroblasts, induced pluripotent stem cells, HepG2, and U87-MG cells. This increase is not caused by improved uptake, but instead appears to be driven by improved intracellular trafficking of polyplexes compared transfection without heparin. Increasing the size of the plasmid cargo from 4.7 kbp to a more therapeutically relevant 10 kbp leads to the complete loss of transfection efficiency in Tr4-heparin transfection of primary fibroblasts and a reduction in transfection efficiency in iPSCs. Co-transfecting with additives meant to increase nuclear localization of the pDNA recovers the efficiency lost by increasing the plasmid size. These techniques allowed for the development of function transfection methods in iPSCs delivering a synthetic transcription activator of collagen type VII. Finally, nanofiber mats containing chondroitin sulfate were developed to scavenge inflammatory molecules from wound exudate.
  • Thumbnail Image
    Item
    Exploring Critical Vehicle Parameters for the Design of Multitargeted Nanoparticles for Cancer Specific Gene Delivery
    (2015-10) Levine, Rachel
    The greatest obstacle to clinical application of cancer gene therapy is lack of effective delivery tools. Gene delivery vehicles must protect against degradation, avoid immunogenic effects and prevent off target delivery which can cause harmful side effects. The stealth liposomes has greatly improved tumor localization of small molecule drugs and is a promising tool for nucleic acid delivery as the polyethylene glycol coating its surface protects against immune recognition and blood clearance. In this study, DNA was fully encapsulated within in stealth liposomes by complexing the macromolecule with a cationic polymer before encapsulation. Formation methods and material compositions were then investigated for their effects on encapsulation. This technology was translated for protective delivery of siRNA designed for HPV viral gene silencing and cervical cancer treatment. Stealth liposomes encapsulating siRNA were functionalized with a targeting peptide which binds to the alpha6 integrin, a cervical cancer biomarker. It was found that both targeting and polymer complexation before encapsulation were critical components to effective transfection. Varying the siRNA:polymer ratio revealed an optimal concentration for enhanced transfection, but no improvement to internalization, suggesting polymer complexation enhances transfection through an intracellular mechanism. Nanoparticles functionalized with cancer-targeting ligands have shown promise but are still limited by off-tumor binding to healthy tissues that nevertheless express low levels of the molecular target. Targeting two unique cancer biomarkers using dual-targeted heteromultivalent nanoparticles presents a solution to this challenge by requiring overexpression of two separate ligands for localization. In order to guide experimental design, a kinetic model was built to explore how the affinity and valency of dual-ligand liposomes affect the binding and selectivity of delivery to cells with various receptor expression. α5β1 and α6β4 integrin expression levels were then quantified on 20 different cell lines to identify appropriate model cells for in vitro investigation. Dual-targeting heteromultivalent liposomes were synthesized using the alpha6 targeting peptide and an alpha5beta1 targeting peptide. Heteromultivalent liposomes with varying peptide ratios were delivered to cells with varying integrin concentrations. Binding and internalization was then evaluated to understand the effect of valency and avidity on binding kinetics and delivery. Dual-ligand liposomes with equal valencies of each targeting peptide achieved enhanced binding efficiency and selectivity for cells expressing equal and high receptor levels. This liposome formulation was used as a gene delivery vehicle to achieve improved transfection to dual-receptor expressing cells. The insights gained from this study inform rational design of modular heteromultivalent nanoparticles for enhanced specificity to target tissue, for the creation of more effective cancer treatments.
  • Thumbnail Image
    Item
    Quinine Copolymer Reporters For Enhanced Gene Editing And Raman Imaging
    (2022-01) Van Bruggen, Craig
    After decades of development, gene therapy has finally reached the forefront of medicine and has led to new cures for genetic disorders and the development of life-saving vaccines. The field has been buoyed by the development of more precise and user-friendly targeted nucleases, such as those used for clustered regularly interspersed palindromic repeats (CRISPR)-based editing. These useful gene-editing technologies, however, are still stymied by the challenge of delivering exogenous nucleic acids and proteins into the cells of interest. The emerging gene therapy industry is investing heavily in developing more efficient and safe non-viral vehicles as alternatives to costly and immunogenic viral vectors. Cationic polymers are promising non-viral vectors due to their manufacturing scalability, their chemical stability, and their synthetic tunability. Improvements in delivery efficiency are necessary, however, for widespread adoption of polymeric vehicles for gene therapy. One challenge in improving performance, however, is the difficulty and limited methodology for elucidating the intracellular mechanics of polymeric vehicles. In this thesis, I describe my research focused on the development of a novel quinine-containing polymer, called a Quinine Copolymer Reporter (QCR), that enhanced transient transfections of cultured cells with plasmids and improved gene editing of cultured cells through the simultaneous delivery of the CRISPR-associated protein Cas9 and DNA donor template. In addition, I describe collaborative research performed with colleagues in the research group of Prof. Renee Frontiera that characterized a band in quinine’s Raman spectrum that is diagnostic of its chemical environment. Using this chemical sensitivity in conjunction with Raman microscopic imaging, we help elucidated the intracellular unpackaging mechanisms of the QCR-nucleic acid complexes.