CRISPR /Cas9‐mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse

Abstract

The X‐linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas‐mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies.

Immunity to CRISPR Cas9 and Cas12a therapeutics

“…In delivering CRISPR‐Cas9 to mouse mammary glands with lentiviruses, one study reported immune cell infiltration that cannot be suppressed by immunosuppression . Another study compared naked DNA hydrodynamic transduction and adenoviral delivery of CRISPR‐Cas9 into hemophilic mice for correcting a transgenic human factor IX mutation . Hydrodynamic plasmid delivery resulted in 0.5–1.5% gene correction that was sufficient to confer the desired blood clotting ability.…”

The CRB1 Complex: Following the Trail of Crumbs to a Feasible Gene Therapy Strategy

“…Despite its growing popularity, however, the CRISPR/Cas9 system is not perfect, as the guide RNA can bind to similar sites outside of the targeted gene, potentially leading to unspecified and unintended mutations, thus limiting both its research value and clinical potential (Fu et al, 2013). Nevertheless, CRISPR/Cas9 has been used to correct defects in several genes, including genes linked to Duchenne muscular dystrophy, metabolic liver disease, and hemophilia B (Guan et al, 2016; Long et al, 2016; Maggio et al, 2016; Nelson et al, 2016; Tabebordbar et al, 2016). Correcting a point mutation requires that the Cas9 protein, guide RNA, and donor template for recombination are introduced together into the same cells.…”

Development and Characterization of MDR1 (Mdr1a/b) CRISPR/Cas9 Knockout Rat Model

“…Compared with ZFN and transcription activator-like effector nuclease (TALEN), the CRISPR/Cas9 system can edit multiple genes simultaneously and has few species limitations Ceasar et al, 2016). At present, CRISPR/Cas9 technology has been widely used in animal modeling Lu et al, 2017), disease research (Maresch et al, 2016), gene therapy (Guan et al, 2016;Wei et al, 2016), and other fields.…”