Browsing by Subject "Minicircle"

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    Enhanced Efficacy of Plasmid DNA Vaccines for Cancer Therapy
    (2010-11) Dietz, Wynette
    Since cancer is the second most common cause of death in the United States, it is of great importance to pursue new and improved methods for treating cancer. The goal of cancer immunotherapy is to exploit the specificity and longevity of immune responses through the use of vaccines to treat cancer. DNA vaccines have many advantages over protein and viral vaccine-based strategies including low cost, ease of production, flexibility and low toxicity. Plasmid DNA vaccines encoding tumor antigens can produce powerful anti-tumor immune responses in animal models, but clinical trials have shown only modest responses. This lack of clinical efficacy is thought to reflect the two major limitations of plasmid DNA vaccines: transient protein expression and low transfection efficiency. Transient protein expression is likely the result of gene silencing due to transcriptionally repressive chromatin within the plasmid backbone. To overcome this limitation, we removed the bacterial backbone sequences and produced a minicircle DNA consisting of the gene expression cassette with only a few bases of the bacterial backbone. This resulted in persistent protein expression, increased transfection efficiency and enhanced immunogenicity. In an effort to further enhance the transfection efficiency, we produced cationic carriers that bind plasmid DNA and protect it from degradation. The addition of these cationic carriers significantly increased transfection efficiency in vitro but has yet to show the same effect in vivo. Additionally, we administered these vaccines transdermally using a tattoo device and achieved rapid and potent immune responses. Our results suggest transdermal delivery of a minicircle DNA vaccine elicits potent antigen-specific immune responses, and as such, holds great promise for cancer therapy. Future work will include increasing transfection efficiency in vivo and increasing the immunogenicity of the vaccines through the use of adjuvants with the goal of producing feasible, efficacious DNA vaccines for cancer therapy.
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    Strategies to improve gene expression and targeting for DNA vaccine development
    (2013-10) Skinner, Nicole Elizabeth
    This dissertation examines strategies for improving DNA vaccines. Despite their many advantages and the considerable promise shown in small animal models, poor immunogenicity resulting from low transfection efficiency and transient transgene expression limits the effectiveness of DNA vaccines in humans. Methods for circumventing transient transgene expression as well as for directly manipulating T cell responses are explored. Chapters 2 and 3 of this thesis study the use of minicircle (MC) DNA as a vaccine. In chapter 2, a MC DNA vaccine is shown to generate larger CD8+ T cell responses and confer superior protection against an infectious challenge than a conventional plasmid DNA vaccine. Chapter 3 investigates the mechanism by which MC DNA elicits superior CD8+ T cell responses. In particular, the role of prolonged antigen expression is explored and found to be an important component of MC DNA vaccine efficacy. Chapter 4 proposes a method for targeted gene delivery to T cells in general, or to antigen-specific T cells. The targeting mechanism uses a streptavidin platform modified with positively charged peptides to enable binding to nucleic acid. Targeting is mediated by addition of biotinylated targeting moieties, either an anti-CD7 antibody for targeting of T cells or peptide-MHC molecules for targeting of antigen-specific T cells. We find that such an approach is promising, as it mediates uptake of both fluorescently labeled siRNA and DNA. However, gene expression did not occur, indicating that further modifications to the approach are needed.