OC 11.2 - In vivo correction of a severe Hemophilia A patient mutation using CRISPR base editing

Background: Hemophilia A (HA) is an inherited bleeding disorder characterized by insufficient production of functional coagulation factor VIII (FVIII). The identification and correction of the patient’s F8 pathogenic variant represents a personalized therapeutic approach to rescue FVIII production and therefore treat HA.

Aims: To use CRISPR cytosine base editing (CBE) to target the HA missense variant to achieve high editing efficiency, resulting in phenotypic and genotypic improvements.

Methods: Among F8 variants identified in our patients with HA, we selected the T535C variant and designed sgRNA to target mutant C·G. To examine the in vivo base editing, we constructed a mutant human FVIII (hFVIII) expression plasmid with the T535C variant and used a wild-type hFVIII plasmid as control. Various CBE plasmids containing either CMV or CAG promoters were examined. T535C mutated FVIII plasmid, along with different CBE plasmids were hydrodynamically injected into an exon 16 deletion-HA murine model. The FVIII activities were determined by activated partial thromboplastin time assay. Genomic DNA was extracted from treated mouse livers, followed by Sanger sequencing to evaluate the editing efficiency.

Results: The CBE variant driven by CAG promoter rescued the highest FVIII activity. The DNA sequencing data showed that ~50% of mutant C·G at site 535 was corrected to the normal T·A, which recovered 220% FVIII activity from mutant FVIII plasmid (0% in mutant hFVIII plasmid, ~460% in wild-type hFVIII plasmid-treated mice) on day 7. The CBE variant plasmid induced a minor bystander effect without significant impact on recovering functional FVIII activity.

Conclusion(s): The results suggest that the base editing tools comprising the CBE variant plasmid and the specific sgRNA can efficiently correct the missense mutation and provide effective phenotypic correction in mutant FVIII plasmid-treated HA mice. We are currently testing the BE tools to correct point mutations in primary human cells.