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All Subjects: nanotechnology

DNA Nanotechnology for Protein Co-Crystallization & Vaccine Delivery

Description

DNA nanotechnology is ideally suited for numerous applications from the crystallization and solution of macromolecular structures to the targeted delivery of therapeutic molecules. The foundational goal of structural DNA nanotechnology was the development of a lattice to host proteins for crystal structure solution. To further progress towards this goal, 36 unique four-armed DNA junctions were designed and crystallized for eventual solution of their 3D structures. While most of these junctions produced macroscale crystals which diffracted successfully, several prevented crystallization. Previous results used a fixed isomer and subsequent investigations adopted an alternate isomer to investigate the impact of these small sequence changes on the stability and structural properties of these crystals. DNA nanotechnology has also shown promise for a variety biomedical applications. In particular, DNA origami has been demonstrated as a promising tool for targeted and efficient delivery of drugs and vaccines due to their programmability and addressability to suit a variety of therapeutic cargo and biological functions. To this end, a previously designed DNA barrel nanostructure with a unique multimerizable pegboard architecture has been constructed and characterized via TEM for later evaluation of its stability under biological conditions for use in the targeted delivery of cargo, including CRISPR-containing adeno-associated viruses (AAVs) and mRNA.

ContributorsHostal, Anna Elizabeth (Author) / Anderson, Karen (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Yan, Hao (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)

Created2021-05

Pathogenic peptides to enhance treatment of glioblastoma: evaluation of RVG-29 from rabies virus and chlorotoxin from scorpion venom

Description

Glioblastoma (GBM) is a highly invasive and deadly late stage tumor that develops from abnormal astrocytes in the brain. With few improvements in treatment over many decades, median patient survival is only 15 months and the 5-year survival rate hovers at 6%. Numerous challenges are encountered in the development of treatments for GBM. The blood-brain barrier (BBB) serves as a primary obstacle due to its innate ability to prevent unwanted molecules, such as most chemotherapeutics, from entering the brain tissue and reaching malignant cells. The GBM cells themselves serve as a second obstacle, having a high level of genetic and phenotypic heterogeneity. This characteristic improves the probability of a population of cells to have resistance to treatment, which ensures the survival of the tumor. Here, the development and testing of two different modes of therapy for treating GBM is described. These therapeutics were enhanced by pathogenic peptides known to improve entry into brain tissue or to bind GBM cells to overcome the BBB and/or tumor cell heterogeneity. The first therapeutic utilizes a small peptide, RVG-29, derived from the rabies virus glycoprotein to improve brain-specific delivery of nanoparticles encapsulated with a small molecule payload. RVG-29-targeted nanoparticles were observed to reach the brain of healthy mice in higher concentrations 2 hours following intravenous injection compared to control particles. However, targeted camptothecin-loaded nanoparticles were not capable of producing significant treatment benefits compared to non-targeted particles in an orthotopic mouse model of GBM. Peptide degradation following injection was shown to be a likely cause for reduced treatment benefit. The second therapeutic utilizes chlorotoxin, a non-toxic 36-amino acid peptide found in the venom of the deathstalker scorpion, expressed as a fusion to antibody fragments to enhance T cell recognition and killing of GBM. This candidate biologic, known as anti-CD3/chlorotoxin (ACDClx) is expressed as an insoluble protein in Nicotiana benthamiana and Escherichia coli and must be purified in denaturing and reducing conditions prior to being refolded. ACDClx was shown to selectively activate T cells only in the presence of GBM cells, providing evidence that further preclinical development of ACDClx as a GBM immunotherapy is warranted.

ContributorsCook, Rebecca Leanne (Author) / Blattman, Joseph N (Thesis advisor) / Sirianni, Rachael W. (Thesis advisor) / Mor, Tsafrir (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)

Created2019

Self-Assembled Nucleic Acid Nanomaterials for Biomedical Applications or Structural Determination of Guest Molecules

Description

Originally conceived as a way to scaffold molecules of interest into three-dimensional (3D) crystalline lattices for X ray crystallography, the field of deoxyribonucleic acid (DNA) nanotechnology has dramatically evolved since its inception. The unique properties of DNA nanostructures have promoted their use not only for X ray crystallography, but for a suite of biomedical applications as well. The work presented in this dissertation focuses on both of these exciting applications in the field: 1) Nucleic acid nanostructures as multifunctional drug and vaccine delivery platforms, and 2) 3D DNA crystals for structure elucidation of scaffolded guest molecules.Chapter 1 illustrates how a wide variety of DNA nanostructures have been developed for the delivery of drugs and vaccine components. However, their applications are limited under physiological conditions due to their lack of stability in low salt environments, susceptibility to enzymatic degradation, and tendency for endosomal entrapment. To address these issues, Chapter 2 describes a PEGylated peptide coating molecule was designed to electrostatically adhere to and protect DNA origami nanostructures and to facilitate their cytosolic delivery by peptide-mediated endosomal escape. The development of this molecule will aid in the use of nucleic acid nanostructures for biomedical purposes, such as the delivery of messenger ribonucleic acid (mRNA) vaccine constructs. To this end, Chapter 3 discusses the fabrication of a structured mRNA nanoparticle for more cost-efficient mRNA vaccine manufacture and proposes a multi-epitope mRNA nanostructure vaccine design for targeting human papillomavirus (HPV) type 16-induced head and neck cancers. DNA nanotechnology was originally envisioned to serve as three-dimensional scaffolds capable of positioning proteins in a rigid array for their structure elucidation by X ray crystallography. Accordingly, Chapter 4 explores design parameters, such as sequence and Holliday junction isomeric forms, for efficient crystallization of 3D DNA lattices. Furthermore, previously published DNA crystal motifs are used to site-specifically position and structurally evaluate minor groove binding molecules with defined occupancies. The results of this study provide significant advancement towards the ultimate goal of the field.

ContributorsHenry, Skylar J.W. (Author) / Stephanopoulos, Nicholas (Thesis advisor) / Anderson, Karen (Thesis advisor) / Blattman, Joseph (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)

Created2023