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Principal Investigator: Renee Nicole Cottle
Organization: CLEMSON UNIVERSITY
Fiscal Year: 2024
Award: $591,640
Funding agency: National Heart Lung and Blood Institute
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.
Terms: <AAV delivered><AAV delivery><AAV-based delivery><AAV-based viral delivery><AAV-mediated delivery><ANGPT5><ANGPTL3><ANGPTL3 gene><APAP><Acetamidophenol><Acetaminophen><Acetominophen><Address><Adeno-Associated Viruses><Adeno-associated-virus-based delivery><Affect><Autologous><Back><Blood Plasma><Blood Precursor Cell><CRISPR><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cancers><Cardiovascular Diseases><Cas nuclease technology><Cell Body><Cell Growth and Maintenance><Cell Maintenance><Cell-Mediated Lympholytic Cells><Cells><Cholesterol><Clinical Trials><Clonal Expansion><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Cytochrome P-450><Cytochrome P-450 Enzyme System><Cytochrome P-450 Oxidase><Cytochrome P-450 Reductase><Cytochrome P450><Cytochrome P450 Family Gene><Cytochrome P450 Reductase><Cytolytic T-Cell><Cytotoxic T Cell><Cytotoxic T-Lymphocytes><Dehydrogenases><Dependoparvovirus><Dependovirus><Disease><Disorder><Dorsum><EC 1.6.2.4><Electroporation><Engineering><Essential Hypercholesterolemia><Familial Hypercholesterolemia><Ferrihemoprotein P-450 Reductase><Ferrihemoprotein P450 Reductase><Fever><Foundations><Genes><Genetic Engineering><Genetic Engineering Biotechnology><Genetic Engineering Molecular Biology><Hematopoietic Progenitor Cells><Hematopoietic stem cells><Hepatic Cells><Hepatic Disorder><Hepatic Parenchymal Cell><Hepatic Transplantation><Hepatocyte><Hereditary><Histocompatibility Complex><Histocompatibility Complices><Human><Hydroxyacetanilide><Hyperbetalipoproteinemia><Hyperlipoproteinemia Type 2><Hyperlipoproteinemia Type II><Immune response><Immunity><Immunological response><Impairment><Inherited><Injury to Liver><Insertional Mutagenesis><Intermediary Metabolism><LDL><LDL Cholesterol><LDL Cholesterol Lipoproteins><LDL Lipoproteins><LDL triglyceride><LDL triglyceride lipoprotein><Liver><Liver Cells><Liver Grafting><Liver Transplant><Liver diseases><Long-Term Effects><Longterm Effects><Low Density Lipoprotein Cholesterol><Low-Density Lipoproteins><Major Histocompatibility Complex><Major Histocompatibility Complices><Malignant Neoplasms><Malignant Tumor><Mediating><Medicine><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Messenger RNA><Metabolic><Metabolic Diseases><Metabolic Disorder><Metabolic Processes><Metabolism><Methods><Mice><Mice Mammals><Modern Man><Murine><Mus><NADPH Cytochrome P-450 Oxidoreductase><NADPH Cytochrome P-450 Reductase><NADPH Cytochrome P450 Oxidoreductase><NADPH Cytochrome c Reductase><NADPH-Cytochrome P450 Reductase><NADPH-Ferrihemoprotein Reductase><NADPH-P450 Reductase><NADPH-Specific Cytochrome C Reductase><Oxidoreductase><Oxidoreductase Gene><P450><Paracetamol><Patients><Peptides><Persons><Plasma><Plasma Serum><Population><Pyrexia><Reagent><Recombinant DNA Technology><Reductases><Research><Resected><Resistance><Reticuloendothelial System, Serum, Plasma><Ribonucleoproteins><Risk><Selection for Treatments><Specificity><Surface Proteins><T cell response><T-Cells><T-Lymphocyte><Testing><Therapeutic><Thesaurismosis><Time><Toxic effect><Toxicities><Transplantation><Triacylglycerol><Triglycerides><Type 2 Hyperlipidemia><Type II Hyperlipidemia><Viral><Work><adeno associated virus group><adeno-associated viral vector delivery><adeno-associated virus delivery><adeno-associated virus mediated delivery><adenovirus mediated delivery><angiopoietin 5><angiopoietin-like 3><beta-Lipoprotein Cholesterol><beta-Lipoproteins><blood cell progenitor><blood progenitor><blood stem cell><blood-forming stem cell><cardiovascular disease risk><cardiovascular disorder><cardiovascular disorder risk><delivered with AAV><delivery with AAV><disease phenotype><electroporative delivery><familial hyperbetalipoproteinemia><familial hypercholesteremia><familial hyperlipoproteinemia type 2><familial hyperlipoproteinemia type II><febrile><febris><gene electrotransfer><genetically engineered><genome editing><genomic editing><genotoxicity><hematopoietic progenitor><hematopoietic stem progenitor cell><hemopoietic progenitor><hemopoietic stem cell><hepatic body system><hepatic damage><hepatic disease><hepatic injury><hepatic organ system><hepatocyte engraftment><hepatopathy><host response><immune system response><immunogenic><immunogenicity><immunoresponse><in vivo><killer T cell><knock-down><knockdown><lipid based nanoparticle><lipid nanoparticle><liver damage><liver disorder><liver injury><liver transplantation><loss of function mutation><low density lipoprotein triglyceride><mRNA><malignancy><metabolism disorder><mouse model><murine model><neoplasm/cancer><new approaches><novel><novel approaches><novel strategies><novel strategy><premature><prematurity><programs><resistant><selection of treatment><therapy selection><thymus derived lymphocyte><transplant><treatment selection><triacylglycerol LDL>