PROJECT SUMMARY
Familial hypercholesterolemia (FH) affects 1 in 250 people and is characterized by impaired low-density
lipoprotein (LDL) metabolism resulting in premature cardiovascular disease. CRISPR-Cas9-induced loss of
function mutations in the gene encoding Angiopoietin-like 3 (ANGPTL3) has been proposed as a therapeutic
strategy to permanently reduce LDL cholesterol and triglyceride levels and lower cardiovascular disease risks.
However, the absence of safe and effective methods for delivering CRISPR components into hepatocytes is a
major barrier. Adeno-associated viruses (AAVs) are the platform of choice for delivering gene-editing reagents
but are associated with severe limitations. In addition, Cas9 immunity is highly prevalent in the human
population further complicating AAV delivery. We propose to introduce gene-editing reagents into hepatocytes
using nonviral delivery approaches ex vivo, then transplanting the engineered hepatocytes to replace diseased
hepatocytes to treat FH. Our nonviral ex vivo strategy avoids two complications of in vivo approaches since
CRISPR-Cas9 editing is only restricted to the intended target cells and enables the opportunity to maintain
cells in culture until they are no longer immunogenic. But this ex vivo approach has one potential drawback:
How to enhance the number of edited hepatocytes engrafted in the liver. To address this challenge, we will use
a novel approach for selecting edited hepatocytes using fever medicine acetaminophen (APAP). We
hypothesize that gene edited hepatocytes lacking NADPH-cytochrome P450 oxidoreductase (Cypor) will be
enriched in vivo by APAP administration without permanent liver damage. In the proposed studies, we will
directly compare LNPs, electroporation and AAVs for gene editing using CRISPR-Cas9 to disrupt Cypor and
Angptl3. We will then replace diseased hepatocytes with gene edited hepatocytes in an established mouse
model of FH (Ldlr–/– ) using APAP selection. In Aim 1, we will compare specificity and efficiency of multiplex
gene editing in Cypor and Angptl3 by electroporation, LNP, and AAV-mediated delivery of CRISPR-Cas9 in
primary mouse hepatocytes. For Aim 2, we will optimize transplantation and APAP-mediated selection of gene-
edited hepatocytes and evaluate the long-term effects of Cypor-knockdown in Ldlr-/- mice while comparing
LNP, electroporation, and AAV-mediated ex vivo delivery of Cypor-CRISPR-Cas9. In Aim 3, we will compare
the effects of nonviral and AAV-mediated multiplex delivery of Cypor and Angptl3-CRISPR-Cas9 on the
capacity of hepatocytes to lower plasma cholesterol and triglyceride levels and circumvent Cas9 immunity in
Ldlr–/– mice subjected to transient APAP treatment. This project is the first to directly compare different nonviral
approaches to AAVs for engineering hepatocytes ex vivo and evaluate the extent that edited hepatocytes
clonally expand in vivo using APAP to treat FH. In addition, this study is the first to study the immunogenicity of
ex vivo gene edited hepatocytes.
