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Principal Investigator: Carol H Miao
Organization: SEATTLE CHILDREN'S HOSPITAL
Fiscal Year: 2024
Award: $758,254
Funding agency: National Heart Lung and Blood Institute
PROJECT SUMMARY
The goal of this project is to combine the technology of lipid nanoparticles (LNPs) and CRISPR/Cas9 gene editing
tools to correct the mutant factor VIII (FVIII) genes and rescue hemophilia A (HemA) phenotype. HemA is a
bleeding disorder resulting from a deficiency of the X-linked FVIII gene. Current treatment of frequent infusions
of FVIII protein is costly, inconvenient, short-term, and incompletely effective. Gene therapy represents a highly
promising alternative method to treat HemA patients. Recent hepatocyte-directed adeno-associated viral (AAV)
gene therapy trials for HemA yielded very promising results, however, FVIII levels dropped precipitously over
time in treated patients due to yet unidentified reasons. Ectopic FVIII expression and misfolding in hepatocytes
may induce cellular stress responses and toxicity. On the other hand, a nonviral in vivo gene editing approach
can provide permanent correction of FVIII gene without using viral vectors. LSECs are the primary natural cellular
source of FVIII biosynthesis. Recently, advancement of biocompatible LNP technology enabled delivery of
nucleic acids safely into target organs. We propose to develop LSEC-targeting LNPs to deliver gene editing tools
for hemophilia treatment. We will synthesize and improve LSEC-targeting LNPs via screening of different lipid
components, optimizing lipid formulations and attaching endothelial-targeting ligands to LNPs, enabling
enhancement of targeting and delivery efficiency into LSECs. In this project, we propose to permanently correct
mutated FVIII gene and regain FVIII expression using a combination of LNPs and Clustered regularly interspaced
short palindromic repeats (CRISPR)/Cas9 endonuclease (Cas9) gene editing tools. We will first investigate
correction of small deletions/insertions in a unique immunodeficient NSG HemA mice. We will use the optimal
LSEC-targeting LNPs to deliver Cas9 mRNA, sgRNAs and DNA templates at different dosages and ratios to
maximize the in vivo gene editing efficacy and examine the correction via indel and/or precision repair. In addition,
in order to facilitate the clinical translation, we will investigate if safe and highly efficient gene editing of small
deletion/insertions derived from HemA patients can be achieved in PBMCs isolated from HemA patients.
Furthermore, we propose to employ the newly developed base editor (BE) to correct single base mutations. BE
can achieve efficient precision editing without double strand breaks (DSBs) for enhanced safety. For selected
point mutations from HemA patients, we will test in vivo gene editing using specific BE in mutant FVIII plasmid
treated HemA mice. Next, we will use the optimal LSEC-targeting LNPs to deliver BE to restore FVIII gene
expression in the corresponding specific transgenic mouse model harboring a human FVIII exon with the
mutation site. Furthermore, we will investigate if the corresponding point mutations can be corrected using the
specific BE in PBMCs isolated from HemA patients. This project will facilitate the development of novel gene
editing strategies for personalized treatment of HemA patients.
Terms: <AAV vector><AAV-based vector><Anabolism><Antihemophilic Factor><Blood Coagulation Disorders><Blood Coagulation Factor VIII><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-nanoparticles><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cells><Cellular Stress><Cellular Stress Response><Cholesterol><Clinical Trials><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 nanoparticles><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Coagulation Disorder><Coagulation Factor VIII><Coagulation Factor VIIIc><Coagulopathy><Complication><DNA><DNA Damage Repair><DNA Repair><DNA Therapy><Data><Deoxyribonucleic Acid><Development><Drops><Encapsulated><Endogenous Factors><Endothelial Cells><Endothelium><Exhibits><Exons><Factor VIII><Factor VIII Deficiency><Factor VIII F8B><Formulation><Gene Delivery><Gene Expression><Gene Transfer Clinical><Gene therapy trial><Genes><Genetic Alteration><Genetic Change><Genetic Intervention><Genetic defect><Genome><Glycans><Goals><Guide RNA><Hemophilia><Hemophilia A><Hepatic Cells><Hepatic Parenchymal Cell><Hepatocyte><Human><In Vitro><In vivo analysis><Infusion><Infusion procedures><Injections><KI mice><Knock-in Mouse><Ligands><Link><Lipids><Liver><Liver Cells><Mediating><Messenger RNA><Methodology><Methods><Mice><Mice Mammals><Modern Man><Murine><Mus><Mutate><Mutation><Non-Polyadenylated RNA><Nucleic Acids><Organ><PBMC><Patients><Peptides><Peripheral Blood Mononuclear Cell><Phenotype><Plasmids><Point Mutation><Polysaccharides><Procoagulant Component><Protein Replacement Therapy><Proteins><RNA><RNA Gene Products><Ribonucleic Acid><Safety><Site><Source><Specificity><System><Technology><Therapeutic Effect><Thromboplastinogen><Time><Toxic effect><Toxicities><Transgenic Mice><Translating><Unscheduled DNA Synthesis><Viral Antibodies><Viral Genes><Viral Vector><adeno-associated viral vector><adeno-associated virus vector><anti-viral antibody><antihemophilic factor A><base><base editor><bases><biocompatibility><biomaterial compatibility><biosynthesis><bleeding disorder><cell stress><clinical applicability><clinical application><clinical translation><clinically translatable><clotting disorder><complex Blood-coagulation factor VIII><cost><design><designing><developmental><dosage><endonuclease><enzyme replacement therapy><experiment><experimental research><experimental study><experiments><gRNA><gene editing method><gene editing methodology><gene editing platform><gene editing strategy><gene editing system><gene editing techniques><gene editing technology><gene editing tools><gene repair therapy><gene therapy><gene transfer trial><gene-based therapy><gene-editing approach><gene-editing toolkit><genetic therapy><genome mutation><genomic therapy><hepatic body system><hepatic organ system><human disease><hypoimmunity><immune deficiency><immunodeficiency><improved><in vivo><in vivo evaluation><in vivo testing><indel><infusions><inhibitor><insertion-deletion><insertion-deletion mutation><insertion/deletion><insertion/deletion mutation><knockin mice><lipid based nanoparticle><lipid nanoparticle><mRNA><mouse model><murine model><mutant><nano medicinal><nano medicine><nanomedicinal><nanomedicine><novel><nucleic acid delivery><personalization of treatment><personalized medicine><personalized therapy><personalized treatment><platelet cofactor I><repair><repaired><screening><screenings><thromboplastinogen A>