Date of Award
Doctor of Philosophy (PhD)
Inherited metabolic diseases (IMDs) of the liver have a collective occurrence of about 1 in 800 births. Current therapies for IMDs of the liver are limited, and the only curative option for patients is orthotopic liver transplantation. Due to shortages of organ donors, immunosuppression following the transplantation, and mortality risks associated with the procedure, alternative treatment options are necessary for curing IMDs of the liver. Therapeutic gene editing has been proposed as a potential strategy for treating IMDs of the liver. Adeno-associated viral vectors (AAVs) are the most widely used delivery method for liver-targeted gene therapies. Although widely used for gene editing, AAV vectors can integrate into the host genome, causing mutagenesis and prompting an immune response. Non-viral methods for delivering gene editing tools would avoid safety risks associated with AAV vectors. This research investigated two methods for delivering CRISPR-Cas9 into primary hepatocytes: electroporation and lipid nanoparticles.
As proof-of-principle for our therapeutic approach, we designed CRISPR-Cas9 targeting hydroxyphenylpyruvate dioxygenase (Hpd), a therapeutic target for hereditary tyrosinemia type I. We delivered Hpd- targeting CRISPR-Cas9 as plasmid DNA, mRNA, and ribonucleoproteins (RNPs) and compared the on and off-target Cas9 editing efficiencies. We achieved high levels of on-target editing in freshly isolated primary mouse hepatocytes. We observed that hepatocytes treated with Cas9 mRNA had reduced on-target editing compared to hepatocytes treated with Cas9 RNP. Human hepatocytes electroporated with Cas9 RNPs also showed high levels of gene editing (>50%). We then compared unmodified and chemically modified sgRNAs and found that in hepatocytes the sgRNA does not need to be chemically modified for Cas9 to achieve high levels of on-target editing.
We also examined the off-target editing in primary mouse hepatocytes electroporated with our Hpd-aiming CRISPR-Cas9. Ten potential off-target sites were identified in the mouse genome with sequences containing up to 3 mismatches to the gRNA sequence. Next-generation sequencing was used to quantify editing at these potential off-target sites. We identified one site (OF3) that had high levels of off-target editing (>20%). We electroporated hepatocytes with a high-fidelity variant of Cas9 (HiFi Cas9) and observed reduced off-target editing.
Next, we delivered CRISPR-Cas9 into hepatocytes by chalcogen-containing lipid nanoparticles (LNPs). We screened different LNP formulations by delivering eGFP mRNA into Hepa 1-6 and HEK293 cells and identified three LNP formulations that provided high transfection efficiency in cells. We next optimized LNP transfection parameters in primary mouse hepatocytes that provided transfection efficiency of up to 45% and cell viability greater than 60%.
Rathbone, Tanner R., "Nonviral Approaches for Delivery of CRISPR-Cas9 Into Hepatocytes for Treatment of Inherited Metabolic Disease" (2022). All Dissertations. 2979.