The CRISPR-Cas9 gene editing technique safely removed the gene expansion that causes Friedrich’s ataxia (FA), allowing for normal frataxin levels and more functional mitochondria in cells taken from patients, an early study reported.
This work, and further experiments in mice, supports the potential of a stem cell approach to treating FA.
The study, “CRISPR-Cas9 Gene Editing of Hematopoietic Stem Cells From Patients With Friedreich’s Ataxia,” was published in the journal Molecular Therapy Methods and Clinical Development.
FA is caused by a mutation in the FXN gene, which codes for frataxin, a protein found in mitochondria — the cells’ energy-producing factories. Specifically, people with FA have an expansion of a GAA repeat in FXN, resulting in lack of frataxin, iron accumulation, and oxidative stress. (G and A in the repeat stand for guanine and adenine, two of the four building blocks of DNA.)
Gene editing offers the possibility of correcting harmful mutations. In the CRISPR-Cas9 technique, a small guide RNA binds to a target DNA sequence and to the Cas9 enzyme. Then, the enzyme cuts the DNA at the targeted location and the cell’s own machinery adds or deletes genetic material.
A research team at University of California at San Diego previously had shown that transplanted hematopoietic stem and progenitor cells (HSPCs) — which can differentiate into other cell types — could prevent muscle degeneration and locomotor deficits, while also improving mitochondrial function, in a mouse model of FA. These benefits required the transfer of frataxin to muscle and muscle cells.
But a transplant carries certain risks, such as the need for immunosuppression and the possibility of graft-vs-host disease. Autologous transplantation, in which patients receive their own cells after being collected for gene editing, could sidestep these concerns.
Using cells from healthy donors, FA patients, and relatives who are carriers of its causal mutation (patients’ parents), the researchers optimized a method for correcting the GAA expansion without undesirable off-target effects.
Initial experiments identified two guide RNAs that removed the GAA repeats in patient-derived cells, restored FXN gene activity, and normalized mitochondrial function.
Although a delay due to a DNA repair mechanism was seen, post-transplant production of blood cells was normal and not associated with toxic effects.
In mice engineered not to mount an immune response against transplanted cells, a high percentage of edited cells were also able to grow into appropriate cell types over three months.
“Our results support the use of the CRISPR-Cas9 to remove the GAA expansion … leading to physiological rescue of frataxin expression, when the percentage of gene editing is sufficiently high, without cytotoxic effects,” the researchers wrote. “This work represents a step toward the clinical translation of autologous transplantation of gene-corrected HSPCs for FRDA [FA].”
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