DefinePK

Abstract

  Hemophilia A is a monogenic bleeding disorder caused by mutations in the F8 gene, resulting in deficient or dysfunctional clotting Factor VIII (FVIII). Conventional therapies rely on frequent FVIII infusions, which are burdensome, expensive, and associated with the development of inhibitory antibodies. This study explores a gene editing-based therapeutic approach using CRISPR-Cas9 to correct F8 mutations in hematopoietic stem cells (HSCs) as a one-time, curative solution. Five single-guide RNAs (sgRNAs) targeting exon 14 of the F8 gene were designed and evaluated for editing efficiency and specificity.Out of all sgRNA4 scored the best compromise between the lowest off-target activity and the highest on-target activity scoring 88.3.  Having high indel rates (up to 49.1%), stable post-editing cell viability (>80%), and reduced off-target activity, sgRNA4 editing was done in HEK293T cells and the patient-derived HSCs.  After in vivo transplantation into NSG hemophagic mice for eight weeks,  The hepatic system-based dominant localization of the vector as confirmed by the immune profiling with minimal increase in the IL-6, TNF-α and IFN-γ was consistent with a favorable safety profile.  By off-target analysis, only one proven site for sgRNA4 was found, which did not have a functional consequence.  These data illustrate the high efficiency, functional improvement and safety profile of CRISpen-Cas9–mediated..