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CRISPR/Cas9 Gene Editing Technology Used to Reverse Inversions Causing Hemophilia A Defect in Patient-Specific Stem Cells; This Success Points Way to Possible Cure

For the first time, chromosomal defects responsible for hemophilia have been corrected in patient-specific induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 nucleases for gene editing. The scientific report of this possibly life-saving advance is titled “Functional Correction of Large Factor VIII Gene Chromosomal Inversions in Hemophilia A Patient-Derived iPSCs Using CRISPR-Cas9.” It published online on July 23, 2015 in Cell Stem Cell. Sufferers of hemophilia live in a perpetual state of stress and anxiety: their joints wear down prematurely and they have bleeding episodes that feel as if they will never end. Their bodies lack the ability to make the clotting factor responsible for the coagulation of blood so any cut or bruise can turn into an emergency without immediate treatment. Hemophilia A occurs in approximately 1 in 5,000 male births and almost half of severe cases are caused by identified “chromosomal inversions.” In a chromosomal inversion, the order of a sequence of base pairs on the chromosome is reversed so that a gene containing the inversion is not expressed properly. In chromosomal inversions in hemophilia A, the sufferer cannot express the blood coagulation factor VIII (F8) gene, and therefore lacks the protein (factor VIII) that causes blood to clot in healthy people. A Korean team, led by Director of the Center for Genome Engineering Dr. Jin-Soo Kim, Institute for Basic Science (IBS), and Professor Dong-Wook Kim at Yonsei University, has experimented with hemophilia-A-patient-derived induced pluripotent stem cells (iPSCs) and hemophilia mice and found a way to correct the hemophilia-A-causioning inversions and repair the clotting factor deficiency that causes hemophilia A. This is believed to be the first time that iPSCs--which possess the ability to change into any cell type in the body—have been used in a procedure like this. The iPSCs were made from urinary cells collected from patients with the chromosomal inversions causing hemophilia A. The team then applied targeted CRISPR/Cas9 nucleases (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) to the iPSCs in an attempt to correct the inversions.

The targeted CRISPR/Cas9 gene editing function reversed the inverted sequence in the F8 genes, which enabled these genes to function correctly. Corrected-iPSCs were induced to differentiate into mature endothelial cells which expressed the F8 gene. These new endothelial cells were able to reverse the F8 deficiency.

To verify that the process worked, the endothelial cells with the inversion-corrected genes were transplanted into F8-deficient mice (mice with hemophilia A) and the mice started producing the F8 clotting factor on their own, which essentially cured them of hemophilia A.

According to Director Jin-Soo Kim, “We used CRISPR RGENs [RNA-guided engineered nucleases] to repair two recurrent, large chromosomal inversions responsible for almost half of all severe hemophilia A cases.”

Professor Dong-Wook Kim added, “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.”

What may be equally as important to the ability to reverse the chromosomal inversion is the fact that there was no evidence of off-target mutations resulting from the correction. This was a precision procedure: only the parts of genome that the team wanted to change were affected.

These findings open the door for further testing and if the results are anything like those obtained in the mice trials, the future of this treatment looks promising.

[Press release] [Cell Stem Cell abstract]