Image from the Salk Institute
Researchers say they have found a way to correct the chromosomal inversions that can cause severe hemophilia A.
The team used CRISPR-Cas9 RNA-guided engineered nucleases (RGENs) to correct the inversions and reverse factor VIII (FVIII) deficiency in patient-specific induced pluripotent stem cells (iPSCs).
Once the iPSCs had matured into endothelial cells, the group transplanted those cells into mice with hemophilia A.
This increased FVIII activity in the mice without any off-target effects.
The researchers described this work in Cell Stem Cell.
“We used CRISPR RGENs to repair two recurrent, large chromosomal inversions responsible for almost half of all severe hemophilia A cases,” said Jin-Soo Kim, PhD, of the Institute for Basic Science in Seoul, Korea.
The inversions involve the FVIII intron 1 homolog (responsible for about 5% of severe hemophilia A cases) and the intron 22 homolog (responsible for about 40% of cases).
The researchers first collected urinary cells from patients with these inversions, using the cells to generate iPSCs. The team then applied CRISPR-Cas9 RGENs.
The gene-editing technique effectively repaired the FVIII gene in iPSCs that had harbored either inversion. The researchers then forced the corrected iPSCs to differentiate into mature endothelial cells and found these cells successfully expressed the FVIII protein.
To verify that the endothelial cells could reverse FVIII deficiency, the team transplanted the cells into mice with hemophilia A.
The mice soon began producing FVIII on their own, although the FVIII activity in these mice was 10% that of wild-type mice. The activity was higher than FVIII activity in control mice with hemophilia A (3.3% of wild-type mice), but the difference was not statistically significant.
“To the best of our knowledge, this report is the first demonstration that chromosomal inversions or other large rearrangements can be corrected using RGENs or any other programmable nuclease in patient iPSCs,” said Dong-Wook Kim, PhD, of Yonsei University College of Medicine in Seoul, Korea.
The researchers also noted that there was no evidence of off-target mutations with the 3 RGENS used in this study.
