Combining two gene editing approaches in cells derived from a patient with severe hemophilia A successfully restored the levels and activity of the faulty clotting factor VIII (FVIII) and reduced bleedings in a mouse model of the disease, according to a new study.
The research, “ssODN-Mediated In-Frame Deletion with CRISPR/Cas9 Restores FVIII Function in Hemophilia A-Patient-Derived iPSCs and ECs,” was published in the journal Molecular Therapy – Nucleic Acids.
Unlike the promising results in clinical trials of hemophilia B, developing a gene therapy for hemophilia A has not been as successful. This has been limited by the inability to package the genetic sequence coding for FVIII — the clotting factor missing in those patients — into commonly used viral delivery vectors. There also is potential risk of adverse immune responses and causing unwanted mutations.
To overcome these limitations, researchers have explored the potential of combining the gene editing CRISPR/Cas9 system with other available strategies, such as using gene editing approaches in pluripotent stem cells — those with the capacity to self-renew and develop into all cells of the adult body.
Another possibility is to use single-strand oligodeoxynucleotide (ssODN) — short DNA molecules used for promoting gene correction. However, the combination of CRISPR/Cas9 system with ssODN has not been fully explored.
A research team at Central South University in China, collected urinary cells from a 21-year-old man with severe hemophilia A, which were reprogrammed into induced pluripotent stem cells (iPSCs). The patient had a mutation in the F8 gene, which generates the FVIII protein, resulting in a smaller version of the protein.
Using CRISPR/Cas9 and ssODN to selectively delete the portion of the F8 gene that was mutated and was causing the disease, the scientists were able to restore the production and activity of FVIII in iPSC-derived endothelial progenitor cells (C-iEPCs). These results were confirmed by culturing the cells in the laboratory, but also when they were transplanted into mice with hemophilia A.
Transplanted mice also experienced significantly fewer bleeding episodes compared to those who did not receive the modified C-iEPCs. Also, these mice showed higher levels of endothelial cells containing the von Willebrand factor, which helps platelets adhere to sites of blood vessel damage and protects FVIII in the circulation, in the liver. Prior work showed that those cells produce normal levels of FVIII after integration in the liver.
“For the first time, we demonstrated in vitro and in vivo the FVIII function that is encoded by the endogenous F8 gene with a partially deleted B domain,” researchers wrote. “Although it addresses only specific cases, this study provides a reference for the repair of other gene mutations.”