Gene Editing for Hemophilia A Treatment Using Lipid Nanoparticles

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.