Development and Delivery of Genome-Editing Therapies for Improved Glioblastoma Treatment
Date of Award
Doctor of Philosophy (PhD)
Biomedical Engineering (ENAS)
Glioblastoma (GBM) is the most common and aggressive type of primary brain cancer. Even with the current standard of care, which consists of surgical resection, radiotherapy, and chemotherapy, GBM patients have a five-year survival rate of only two percent. Several characteristics of GBM hinder the development of effective treatments. First, GBM is highly invasive by nature, making complete surgical resection of the tumor impossible and tumor recurrence inevitable. Second, a subpopulation of cells within GBM, known as cancer stem cells (CSCs), significantly contributes to patient mortality. CSCs are capable of tumor initiation, invasion, and self-renewal, and are resistant to conventional radiotherapy and chemotherapy. Third, because GBM is located behind the blood-brain barrier, delivery of therapeutics to the tumor site is a non-trivial task. To address these shortcomings, this dissertation aims to (1) elucidate cellular and molecular mechanisms of GBM invasion, (2) identify and validate genetic regulators of CSCs as targets for CSC-directed GBM therapies, and (3) develop a novel delivery system capable of gene editing in the brain. First, to better understand GBM invasion, we performed integrative analysis of histological and single-cell RNA sequencing data from ten GBM patients. At the cellular level, we found that invasive GBM are less dense and less proliferative than their non-invasive counterparts. We show that while CSCs correlate with GBM invasion, only the CD44-expressing subpopulation significantly contributes to the invasive phenotype. At the molecular level, we found that GBM invasion is driven by distinct genes (ADM, S100B, CD44), regulons (EGR1, JUN, FOSB), and signaling pathways (Rho GTPase, integrin, interleukin signaling). We identify CRYAB as a major driver of GBM invasion and demonstrate that CRYAB correlates with CD44 expression and contributes to post-operative recurrence. Taken together, this work establishes a cellular and molecular landscape of GBM invasion that can guide the development of more effective therapies. Next, to identify targets for CSC-directed GBM therapies, we performed single-cell trajectory analysis, in combination with an image-based genome-wide RNAi screen, on patient-derived GBM neurospheres. We identify High Mobility Group Box 2 (HMGB2) and Zinc Finger Protein 117 (ZNF117) as regulators of CSCs in GBM and show that knockout of either gene significantly reduces stemness and tumorgenicity of CSCs in vitro. We demonstrate that HMGB2 may regulate CSC survival through DNA damage response pathways and that ZNF117 controls CSC differentiation towards the oligodendroglial lineage via the Notch pathway through interaction with JAG2. Furthermore, CRISPR/Cas9-mediated knockout of HMGB2 or ZNF117 significantly prolongs overall survival in GBM mouse xenograft models. Ultimately, these findings provide new targets for CSC-directed GBM therapies and have the potential to improve tumor response to current treatments. Finally, to design a programmable delivery system capable of performing gene editing in the brain, we synthesized and screened a library of solid modified poly(amine-co-ester) terpolymer nanoparticles. We demonstrate that modifying the terpolymer chain with chemical moieties that improve cell penetration, endosomal escape, or DNA release significantly enhances gene transfection. Additionally, we found that redox-responsive and acid-responsive terpolymer nanoparticles are capable of transfecting cells significantly greater than that of the commercial transfection reagent Lipofectamine 2000. Lastly, we delivered CRISPR/Cas9-editing cargo using the lead terpolymer nanoparticles to a HEK293 traffic light reporter system. We found that these nanoparticles are capable of performing CRISPR/Cas9-editing with excellent efficiency higher than that of Lipofectamine 2000. In summary, this work describes a simple and effective delivery system that has excellent gene transfection capabilities and translational potential for treatment of disorders in the brain. Taken together, this dissertation offers new insights into biological mechanisms that drive GBM invasion, identifies novel regulators of CSCs in GBM, and describes a new delivery system that can efficiently perform gene editing in the brain. These findings provide complementary strategies to build upon the efficacy of current GBM treatments and have the potential to significantly improve GBM patient outcomes.
Chen, Ann Tai, "Development and Delivery of Genome-Editing Therapies for Improved Glioblastoma Treatment" (2022). Yale Graduate School of Arts and Sciences Dissertations. 463.