Targeted drug delivery to leukemia cells using DNA origami

An estimated 600,000 Americans will die from cancer in 2017. Acute myeloid leukemia (AML) is a particularly deadly form of cancer, with a five-year survival rate of only 26 percent. Chemotherapy, currently the frontline treatment method, has two main limitations: cancer cells can develop drug resistances and the drugs harm healthy cells as well as cancer cells. Currently no FDA-approved targeted therapies exist for AML. The purpose of this project is to develop an effective nanoparticle based cancer drug delivery device that can target and destroy AML cells. DNA nanostructures have recently garnered attention as a novel method for cancer drug delivery due to the precise control they allow over nanostructure geometry. It has recently been shown that when daunorubicin, a drug widely used to treat AML, is attached to DNA nanostructures, the nanostructures allow the drug to circumvent developed drug resistance in cancer cells. Therefore, we hypothesize that building a targeted version of these DNA drug delivery devices could lead to an effective treatment for AML and other cancers. We developed a method that uses the specificity of antibody-antigen interactions to use DNA origami to target CD33 antigens on HL-60 AML cells. We utilized the highly precise geometry of DNA origami to attach anti-CD33 antibodies to specific locations on a nanostructure, to prevent non-target cell interactions. Fluorescent microscopy experiments showed that when the anti-CD33 antibody is attached to a DNA nanostructure, without cancer drugs and in the presence of other cell types, the nanostructure preferentially bound to the cell membranes of HL-60 AML cells. Drug-loaded experiments have not yet shown conclusive evidence, and more experiments need to be performed to ascertain if the drug affects targeting. We believe the drug may not be attached strongly enough to the nanostructure and the design may need to be altered. However, the promising targeting results we have obtained suggest DNA origami has strong potential as a cancer drug delivery device that can potentially simultaneously circumvent drug resistance and target cancer cells.A five-year embargo was granted for this item.Academic Major: Mechanical Engineerin

Itk Negatively Regulates Induction of T Cell Proliferation by CD28 Costimulation

CD28 is a cell surface molecule that mediates a costimulatory signal crucial for T cell proliferation and lymphokine production. The signal transduction mechanisms of CD28 are not well understood. Itk, a nonreceptor protein tyrosine kinase specifically expressed in T cells and mast cells, has been implicated in the CD28 signaling pathway because of reports that it becomes phosphorylated on tyrosines and associates with CD28 upon cross-linking of the cell surface molecule. To determine whether Itk plays a functional role in CD28 signaling, we compared T cells from Itk-deficient mice and control mice for their responses to CD28 costimulation. T cells defective in Itk were found to be fully competent to respond to costimulation. Whereas the CD3-mediated proliferative response was severely compromised in the absence of Itk, the calcineurin-independent CD28-mediated response was significantly elevated when compared with cells from control animals. The augmented proliferation was not due to increased production of interleukin-2. The results suggest that Itk has distinct roles in the CD3 versus the CD28 signaling pathways. By negatively regulating the amplitude of signaling upon CD28 costimulation, Itk may provide a means for modulating the outcome of T cell activation during development and during antigen-driven immune responses

Conformational snapshots of Tec kinases during signaling

The control of cellular signaling cascades is of utmost importance in regulating the immune response. Exquisitely precise protein-protein interactions and chemical modification of substrates by enzymatic catalysis are the fundamental components of the signals that alert immune cells to the presence of a foreign antigen. In particular, the phosphorylation events induced by protein kinase activity must be spatially and temporally regulated by specific interactions to maintain a normal and effective immune response. High resolution structures of many protein kinases along with supporting biochemical data are providing significant insight into the intricate regulatory mechanisms responsible for controlling cellular signaling. The Tec family kinases are immunologically important kinases for which regulatory details are beginning to emerge. This review focuses on bringing together structural insights gained over the years to develop an understanding of how domain interactions both within the Tec kinases and between the Tec kinases and other signaling molecules control immune cell function

Steady-State, Zero Growth and the Academic Library (Book Review)

published or submitted for publicatio