Targeted Delivery of Gene Therapy for Treatment of Neurofibromatosis
Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biomedical Engineering (ENAS)
First Advisor
Zhou, Jiangbing
Abstract
Neurological disorders stand as the leading cause of disability worldwide, with their contribution to the overall burden of health conditions still increasing. The primary drivers of this burden include the limited pharmaceuticals designed for treating the nervous system, especially the brain. As various therapeutic avenues are explored, gene therapy emerges as a promising solution, addressing the root causes of genetic disorders by introducing functional genes or correcting defective ones. Gene therapies typically involve the use of DNA, mRNA, and genome editing system, commonly delivered into the host genome through viral vectors. Due to the limitations in size and safety concerns associated with viral vectors, non-viral vectors offer an alternative approach to overcome these challenges. The primary objectives of this study are to develop and characterize non-viral machinery for the delivery of nucleic acids and the CRISPR/Cas system for gene therapy. Furthermore, the optimized non-viral machinery is assessed in neurological disease models, particularly neurofibromatosis type I (NF1) and type II (NF2) models for effective disease treatment. One of the key non-viral delivery vehicles explored in this study is the use of nanoparticles (NPs). A thorough screening process through a luciferase reporter assay, involving over 200 polymers, identified top stimuli-responsive NPs with efficacy surpassing commercial transfection reagents. These NPs demonstrated exceptional gene transfection capabilities, including DNA and mRNA delivery, showing adaptability in both in vitro and in vivo applications. The NPs were successfully applied to genome editing by delivering CRISPR/Cas9 and achieving successful genome editing in the traffic light reporter system. Another significant non-viral delivery system investigated is the modified ribonucleoproteins (RNPs). Engineered stimuli-responsive RNPs displayed remarkable genome editing efficacy. Through convection-enhanced delivery injections into the brains of Ai9 reporter mice, the RNPs showed editing success in tdTomato expression along the injection tract, emphasizing their adaptability and effectiveness in vivo. Evaluation of RNP concentrations revealed a balance between dispersion, editing efficiency, and potential toxicity, emphasizing the need for optimization. However, no detectable toxicity was observed in circulation, highlighting the safety of the stimuli-responsive RNP delivery system through the route of systemic administration. The study further examined into the introduction of cholesterol (CLS) onto the modified RNPs, resulting in a substantial enhancement in efficiency. Various CLS-RNP formulations were explored, demonstrating promising genome editing efficiencies in Ai9 reporter cells and increased dispersion in Ai9 reporter mice. This development opens new avenues for enhancing the efficacy of genome editing, revealing the therapeutic potential of the engineered CRISPR/Cas9 delivery vehicles. A third unique delivery system, the autoantibody, was also explored in the study. The anti-DNA autoantibody 4H2, initially identified in lupus, has shown to be a candidate as a gene delivery vehicle that could provide a synergistic therapeutic approach through gene therapy and immunotherapy. With our recent studies introducing the concept of 4H2 for cancer immunotherapy, we further explored its potential in gene delivery. The unique properties of the 4H2 autoantibody in transporting nucleic acids, penetrating cells, and avoiding endosomes were investigated. The findings revealed that 4H2 effectively carries DNA into cells, achieves endosomal escape, and demonstrates efficiency in treating genetic diseases. Examining the potential of 4H2 in treating neurological diseases, we employed NF1 and NF2 sciatic nerve tumor models. Treating NF1 and NF2 xenografts with the 4H2/DNA complexes suggested a decrease in tumor burden, emphasizing the adaptability of 4H2 as a carrier for nucleic acids, offering a synergistic approach for gene therapy in addition to immunotherapy. In conclusion, this study presents the development of three exceptional non-viral vehicles for targeted gene therapy and their application for treatment of neurofibromatosis. From efficient gene delivery using nanoparticles to genome editing with modified RNPs and the unique capabilities of autoantibody 4H2, the findings offer insights for translational applications in treating neurofibromatosis, opening new avenues as a potential for treating other neurological disorders.
Recommended Citation
Sheu, Wendy C W, "Targeted Delivery of Gene Therapy for Treatment of Neurofibromatosis" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1387.
https://elischolar.library.yale.edu/gsas_dissertations/1387
