Date of Award
Chemistry and Biochemistry
David Sabatino, Ph.D.
Wyatt Murphy, Ph.D.
James Hanson, Ph.D.
Stephen Kelty, Ph.D.
siRNA nanostructures, Theranostics, Cancer gene therapy, Glucose Regulated Proteins (GRPs), Fluorescence, Prostate cancer
Gene therapy has emerged as a promising precision nano-medicine strategy in the treatment of numerous diseases including cancer. At the forefront of its utility are the applications of short-interfering RNA (siRNA), that silence oncogenic mRNA expression leading to cancer cell death through the RNA interference (RNAi) pathway. Despite the therapeutic potential, siRNAs are limited by poor pharmacological properties, which has hindered their translation into the clinic. Recent studies, however, have highlighted the applications of modified siRNAs, including the use of fluorescent probes and siRNA nanostructures in cancer detection and treatment. The siRNAs reported in this thesis are designed to target and silence the overexpression of the Glucose Regulated Proteins (GRPs) in cancer. The GRPs are a class of chaperone proteins involved in protein folding events within the endoplasmic reticulum. Under physiological or pathological stress, some GRPs translocate to the cell surface where they function as signaling receptors for oncogenic activity. Therefore, cancer cell surface GRPs have been classified as clinically proven biomarkers.
Chapter 2 outlines the design, synthesis and characterization of linear as well as novel V- and Y-shape RNA templates with the use of a ribouridine branchpoint synthon. The RNA templates self-assembled into spheres, triangles, squares, pentagons and hexagons of discrete sizes and shapes, confirmed by native PAGE while TEM imaging validated the sizes and shapes of the siRNA nanostructures. Moreover, thermal denaturation and CD spectroscopy were used to ascertain the prerequisite siRNA hybrids for their RNAi applications. Of interest from this initial study, the self-assembled siRNA hybrids (5 nM) that targeted GRP-75, 78 and 95 elicited a synergistic effect, which resulted in potent gene knockdown as well as cancer cell death.
The simultaneous integration of therapy and diagnostics (“theranostics”) has been utilized to diagnose and treat cancers at their earliest stages, when they are most likely curable or at least treatable. Building on the work described in Chapter 2, the covalently attachment of fluorescein isothiocyanate (FITC) allowed for monitoring cell uptake and biological activity of a wide range of siRNA motifs. Incorporation of FITC had negligible influence on the A-type helix of the siRNAs, while maintaining good hybrid thermal stability. The FL-siRNAs showed some degree of fluorescence quenching relative to the unlabeled siRNAs. However, the higher-order V- and Y-shape siRNA structures enabled the incorporation of multiple fluorescent reporters, which increased siRNA fluorescence but failed to overcome the quenching effects. Upon transfection within the PC-3 prostate cancer cells, the FL-siRNA hybrids (50 nM) exhibited less mRNA knockdown (~10-30%) when compared to their non-labeled counterparts (~40-80%). Conjugation of the FITC probe to the sense strand of the siRNAs re-established mRNA knockdown (~50-
Kozuch, Stephen David, "Fluorescently Labeled siRNAs and their Theranostic Applications in Cancer Gene Therapy" (2018). Seton Hall University Dissertations and Theses (ETDs). 2571.