Date of Award
Doctor of Philosophy (PhD)
Inherited metabolic diseases (IMDs) affecting the liver are relatively rare but collectively have a prevalence of 1 in 800 live births. These diseases result from autosomal recessive single-gene mutations, leading to organ dysfunction and potentially fatal consequences if left untreated. One potential therapeutic strategy for IMDs of the liver involves using CRISPR-Cas9-induced loss of function mutations. However, translating this approach into the clinic is limited by the need for safe and effective CRISPR delivery methods. Adeno-associated viral vectors (AAVs), commonly used for CRISPR delivery, are associated with significant safety and efficacy concerns, including risks for immunogenicity, off-target mutagenesis, and genotoxicity due to persistent Cas9 expression. Therefore, alternative delivery approaches are needed. In this study, we explore the safety and efficacy of electroporation as an alternative to viral delivery methods for ex vivo gene editing therapy for IMDs of the liver. Our strategy involves isolating hepatocytes from the resected liver of a patient, delivering CRISPR components into the hepatocytes via electroporation ex vivo, and subsequently transplanting the edited hepatocytes back into the patient's liver to replace the native hepatocytes with healthy gene-edited ones. Compared to systemic delivery, this approach offers advantages such as cell-specific editing, the potential to screen for off-target effects, and the opportunity to expand or cryopreserve gene-edited hepatocytes for transplantation when needed. The success of a cell-based therapy will require the edited hepatocytes to engraft and replace approximately 5-10% of the liver mass and persist for the patient's lifetime. iii We develop technical protocols to advance a cell-based therapeutic approach. First, we optimize hepatocyte isolation protocols to achieve high viability in freshly isolated primary hepatocytes for downstream delivery. Second, we established a mouse model for primary hepatocyte isolation and transplantation. After that, we show a successful cell therapy approach to reprogram metabolic pathways by electroporating CRISPR-Cas9 mRNA and RNPs to disrupt the gene encoding 4-hydroxyphenylpyruvate dioxygenase (Hpd) in primary hepatocytes ex vivo, followed by transplantation to treat a mouse model of hereditary tyrosinemia type 1 (HT1). Third, to assess the broader applicability of our strategy, we explore a novel cell-based gene editing approach in a mouse model of familial hypercholesterolemia. Specifically, we demonstrate clonal expansion and liver repopulation by gene-edited hepatocytes after transplantation by transient administration of acetaminophen. This study aims to provide valuable insights into the efficacy and safety of electroporation as a nonviral delivery approach for cell-based gene editing therapies for liver disease. These advancements can be applied to many inherited metabolic liver disorders and are a step toward autologous cell therapy in the liver.
Ates, Ilayda, "Investigating the Potential of a Cell-Based Gene Editing Therapy for Inherited Metabolic Liver Disease" (2023). All Dissertations. 3415.