CRISPR-Cas9 and targeted sgRNAs can revert large inversions in hemophilia A iPSCs •Endothelial cells derived from corrected iPSCs express correctly spliced Factor VIII •Transplantation of corrected iPSCs can rescue injury mortality in hemophiliac mice •Whole-genome and targeted deep sequencing did not detect off-target mutations Credit: Chul-Yong Park, Duk Hyoung Kim, Jeong Sang Son, Jin Jea Sung, Jaehun Lee, Sangsu Bae, Jong-Hoon Kim, Dong-Wook Kim, Jin-Soo Kim. Functional Correction of Large Factor VIII Gene Chromosomal Inversions in Hemophilia A Patient-Derived iPSCs Using CRISPR-Cas9. Cell Stem Cell, 2015; DOI:
Hemophilia A occurs in about 1 in 5,000 male births and almost half of severe cases are caused by identified “chromosomal inversions” involving introns 1 and 22 of the F8 gene ie base pair order on chromosome is reversed so the gene doesn’t express properly and the sufferer lacks the blood coagulation factor VIII (F8) gene, which causes blood to clot in healthy people. Their joints wear down prematurely and bleeding episodes feel like they will never end.
This was the 1st time iPSCs, which possess the ability to change into any cell type in the body, was used in a procedure like this. The urinary cells were collected from patients with the chromosomal inversions causing hemophilia to make iPSCs, the team applied CRISPR-Cas9 nucleases (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9) to them.
METHOD: CRISPR-Cas9 reverted the F8 genes which enabled them to function correctly. Corrected-iPSCs were induced to differentiate into mature endothelial cells which expressed the F8 gene. These new endothelial cells reversed the F8 deficiency >> transplanted into F8 deficient mice (mice with hemophilia A) and the mice started producing the F8 clotting factor on their own, which cured them of hemophilia A.
Director Jin-Soo Kim, “We used CRISPR RGENs [RNA-guided engineered nucleases] to repair 2 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.”
+ 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. http://www.alphagalileo.org/ViewItem.aspx?ItemId=154913&CultureCode=en
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