Abstract Number: PB1156
Meeting: ISTH 2020 Congress
Theme: Hemophilia and Rare Bleeding Disorders » Novel Biotherapeutics in Hemophilia
Background: The current treatment for hemophilia A (HA) patients is FVIII protein replacement. However, it needs multi-dose injections which is costly and inconvenient. CRISPR/Cas9 is used to perform gene editing in vitro and in vivo. Correction of HA patients’ nonsense mutation by gene editing can regain expression of missing protein permanently. Furthermore, advancement of nanoparticle (NP) technology can provide a biocompatible and gene delivery system. We investigate if NP technology can achieve targeted gene correction in a HA mouse model.
Aims: To examine gene delivery efficiency of NP in vitro and regain FVIII function in HA mice using CRISPR/Cas9 in vivo.
Methods: NPs carrying p2X-GFP plasmid was examined for transfection efficiency in HUVEC cells by flow cytometry. Immunodeficient hemophilia A mice (NSG HA) with indel mutation in exon 1 of FVIII gene were used as an animal model. sgRNAs that target mouse FVIII gene (mF8sgRNA) or mutant FVIII gene in NSG HA mice (NSGHAsgRNA) were examined in vitro using T7E1 assay, respectively. Mice that were hydrodynamically injected with sgRNA and Ca9 protein expressing plasmids simultaneously. Blood was collected periodically to examine FVIII activity in plasma by aPTT assay.
Results: DNA electrophoresis showed that NPs can carry plasmid efficiently. Flow analysis suggested that NPs carrying p2X-GFP can efficiently transfect HUVEC cells compared to control. mF8sgRNA but not NSGHAsgRNA can specifically induce indel mutation in NIH3T3 cells. NSG HA mice challenged with hydrodynamic injection of mF8sgRNA or NSGHAsgRNA regained FVIII activity at day 7.
Conclusions: NPs can efficiently transfect endothelial cells as shown by GFP expression in HUVEC cells. Both mF8sgRNA and NSGHAsgRNA can induce indel mutation in frameshift site of NSG HA mice, leading to therapeutic levels of FVIII expression. Combining NP and gene editing technology has the potential to recover FVIII gene expression in HA patients and rescue them from daily infusion of recombinant proteins.
[Figure 1. Transfection efficiency of NPs in HUVEC cells]
[Figure 2. Regained FVIII activity in NSG HA mice after gene editing]
To cite this abstract in AMA style:
Chen C-, Cai X, Miao C. Rescue of the Endogenous FVIII Expression in Hemophilia A Mice Using CRISPR/Cas9 Gene Editing [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/rescue-of-the-endogenous-fviii-expression-in-hemophilia-a-mice-using-crispr-cas9-gene-editing/. Accessed September 27, 2023.« Back to ISTH 2020 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/rescue-of-the-endogenous-fviii-expression-in-hemophilia-a-mice-using-crispr-cas9-gene-editing/