Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations

Abstract

Factor V deficiency, an ultra-rare congenital coagulopathy, is characterized by bleeding episodes that may be more or less intense as a function of the levels of coagulation factor activity present in plasma. Fresh-frozen plasma, often used to treat patients with factor V deficiency, is a scarcely effective palliative therapy with no specificity to the disease. CRISPR/Cas9-mediated gene editing, following precise deletion by non-homologous end-joining, has proven to be highly effective for modeling on a HepG2 cell line a mutation similar to the one detected in the factor V-deficient patient analyzed in this study, thus simulating the pathological phenotype. Additional CRISPR/Cas9-driven non-homologous end-joining precision deletion steps allowed correction of 41% of the factor V gene mutated cells, giving rise to a newly developed functional protein. Taking into account the plasma concentrations corresponding to the different levels of severity of factor V deficiency, it may be argued that the correction achieved in this study could, in ideal conditions, be sufficient to turn a severe phenotype into a mild or asymptomatic one.

Keywords: CRISPR; coagulopathies; factor V deficiency; gene editing; gene therapy; rare diseases.

Figure 1

Gene editing strategy. (A) Structure of coagulation factor V. (B) KO HepG2 model development, simulating the similar mutation exhibited by the patient. (C) Patient’s Thr1093* pathological mutation. (D) Correction of the pathological mutation to obtain a treated HepG2 cell line (TT HepG2). (E) End-point PCR for the fragment of the F5 gene on the wildtype HepG2 cell line, the three KO HepG2 clones (LO1, LO8 and LO9) and TT HepG2 (LO1, LO8 and LO9). A 100 bp FastGene molecular marker from Nippon genetics (Japan) was used.

Figure 2

Sequencing of the different editing procedures performed in the F5 of the HepG2 cell line. (A) Editing efficiency of the WT HepG2 line for obtaining the KO HepG2 line (by deleting 35 bp). (B) Three clone KO HepG2 cell lines (LO1, LO8 and LO9) were homozygous for the deletion of 35 bp, giving rise to the formation of a codon stop. (C) Editing efficiency of the KO clones for canceling the mutation and restoring the gene’s frameshift, giving rise to a corrected TT HepG2 cell line. Indel: insertion/deletion. Analyses were developed with the online version of Synthego’s ICE software.

Figure 3

Characterization of different HepG2 lines. (A) Morphological changes of different clones (1, 2 and 3) (scale bar, 200 μm). Representative images from different experiments. (B) Proliferation curve presented as mean ± SD (n = 4) (p < 0.05). (C) Flow cytometry for membrane markers: Unstained cells (in red), WT HepG2 (in blue), KO HepG2 (in green) and TT HepG2 (in orange). Representative images from three different experiments.

Figure 4

Coagulation factor V immuno-staining of cell lines. (A) WT HepG2. (B) KO HepG2. (C) TT HepG2. (D) hASCs as negative control (scale bar, 50 μm). Representative images from three different experiments.

Figure 5

Application of the Human Coagulation Factor V ELISA assay to different HepG2 cell lines (WT HepG2, KO HepG2 and TT HepG2). Control (−) Oval cells and SW480 as negative controls. Figure presented as mean ± SD (n = 3).

Figure 6

Functional factor V coagulometry assay. Functional factor V secreted by different cell lines (WT HepG2, KO HepG2 and TT HepG2). Control (-): Oval cells and SW480 as negative controls. Figure presented as mean ± SD (n = 4) (*** p < 0.001; ** p < 0.01).

Figure 7

Summary of strategies and perspectives of the treatment of factor V deficiency. Using CRISPR/Cas9-based gene editing, the FV-producing HepG2 cell line was modified, giving rise to an in vitro knockout cell model unable to produce functional FV. The same tool was used to treat the modified cells and thereby correct the resulting mutation. This led to the recovery of 41% of FV’s functionality. This technology could constitute a gene therapy that could potentially cure FV deficiency.