OC 12.3 - Rescue of the endogenous FVIII expression in hemophilia A mice using CRISPR/Cas9 mRNA LNPs

Background: Correction of hemophilia A (HA) patients’ nonsense mutation by gene editing can regain expression of missing protein permanently. Furthermore, lipid nanoparticle (LNP) technology can provide an efficient gene delivery system. We investigate if LNP technology combined with gene-editing tools using CRISPR/Cas9 can achieve targeted gene correction in a specific HA mouse model.

Aims: To examine gene delivery efficiency of LNP in vitro and regain FVIII function in HA mice using CRISPR/Cas9 mRNA LNPs in vivo.

Methods: LNPs carrying luciferase mRNA were examined for transfection efficiency in HepG2 and HUVEC cells. The efficiency of two sgRNAs was examined in vitro using a T7E1 assay. In vivo transfection efficiency was investigated by immunofluorescent staining of the liver following intravenous injection of GFP mRNA LNPs into the mice. Immunodeficient HA mice (NSG HA) with indel mutation in FVIII exon 1 were used as a gene-editing animal model. NSG HA Mice were hydrodynamically injected with FVIII exon 1 targeting sgRNA/Cas9 expressing plasmid. Subsequently, CRISPR/Cas9 mRNA LNPs were synthesized and intravenously injected into NSG HA mice. FVIII activity was examined by aPTT assay and gene editing was verified by DNA sequencing.

Results: The data suggested that mRNA LNPs can efficiently transfect both HepG2 and HUVEC cells, and can be delivered to hepatocytes and liver sinusoidal endothelial cells (LSECs) in vivo. NSG HA mice treated with a hydrodynamic injection of sgRNA/Cas9 plasmids regained FVIII expression. . Subsequently, CRISPR/Cas9 mRNA LNPs delivered into NSG HA mice successfully induced indel mutation in mutant FVIII gene and rescued endogenous FVIII activity.

Conclusion(s): Our LNPs can transfect both hepatocytes and LSECs in vitro and in vivo. LNP carrying CRISPR/Cas9 mRNA and mF8sgRNA can induce indel mutation in the target site, leading to therapeutic levels of FVIII expression. These technologies have the potential to repair FVIII mutations and recover FVIII gene expression in HA patients.