Fanconi anemia is a rare genetic disease in which essential DNA repair pathway genes are mutated, disrupting the DNA damage response. Patients with Fanconi anemia experience hematological complications, including bone marrow failure, and are predisposed to cancer. The only curative therapy for the hematological symptoms of Fanconi anemia is an allogeneic hematopoietic stem cell transplant, in which a patient receives healthy stem cells from a donor. While this may cure or prevent some of the disease’s complications, stem cell transplantation can cause additional difficulties, including graft-versus-host disease (GvHD) and exacerbated cancer risk.1
There is growing interest in applying genome editing technologies like CRISPR-Cas9 to correct Fanconi anemia mutations in patient-derived cells for autologous transplants, in which corrected stem cells are given back to the patient. However, this disease poses a unique challenge: How do you apply a genome editing technique in cells that are particularly sensitive to DNA damage? Fanconi anemia cells cannot resolve the double-strand breaks that conventional CRISPR-Cas9 gene editing creates in the target DNA, which prevents researchers from effectively correcting disease-causing mutations with this method.
In a study published in International Journal of Molecular Science, a research team at the University of Minnesota led by Branden Moriarity and Beau Webber used Cas9-based tools called base editors (BEs) to edit genes in Fanconi anemia patient-derived cells without inducing double-strand DNA damage.2 BEs are fusion proteins made of a Cas9 enzyme that cleaves target DNA (nCas9) and a deaminase that converts cytidine to uridine (cytosine base editor, CBE) or adenosine to inosine (adenosine base editor, ABE). During DNA replication or repair, sites targeted by a BE are rewritten as thymine in the case of CBEs, or guanine with ABEs.
Although base editors do not induce double-strand breaks, they still nick the DNA and trigger a DNA repair response. Because of this, the researchers first examined if CBEs and ABEs would work on non-Fanconi anemia genes in patient-derived cells. “There was that mystery, you know, because [Fanconi anemia patient cells are] DNA repair deficient. So we weren't sure…we thought maybe it would work, but not as well as a normal cell. But indeed, it works on the same level, basically. So that was pretty exciting,” Moriarity explained.
The research team then demonstrated that CBEs and ABEs can correct Fanconi anemia-causing mutations in the FANCA gene in primary patient fibroblast and lymphoblastoid cell lines. Base editing restored FANCA protein expression and improved the ability of the patient-derived cells to grow in the presence of a DNA damaging chemical. Additionally, in culture, fibroblasts with corrected FANCA mutations outgrew cells in which the base editing failed. Finally, the researchers assessed if BEs could correct mutations in different Fanconi anemia genes. Using an algorithm, they predicted that most Fanconi anemia mutations were correctable either by BEs or by another nCas9-fusion technology called prime editing (PE), which is capable of large genetic insertions and deletions.
This work comes on the heels of a preprint from another research group at The Centre for Energy, Environmental and Technological Research and ETH Zurich, who investigated ABEs in patient blood cell lines. This group also effectively targeted Fanconi anemia genes with BE technology, and their investigation went one step further: they corrected mutations in patient-derived hematopoietic stem cells.3 This was something that Moriarity and Webber were unable to do—because the disease is a bone marrow failure syndrome, these cells are scarce. “Basically, these patients do not have stem cells,” explains Annarita Miccio, a senior researcher and lab director at Institute Imagine of Paris Cité University, who was not involved in either study. “These are very challenging experiments, and more than the experiments, the challenge of [treating] Fanconi anemia is exactly that—the number of cells.”
Despite this challenge, the researchers have laid the groundwork for genome editing as a treatment approach in Fanconi anemia, without the need for double-strand DNA breaks. “I think the study we did is a good, solid proof of concept, and sets the stage for the next steps, but certainly, it's not the end of the story,” said Webber.
- B.P. Alter, “Inherited bone marrow failure syndromes: considerations pre- and posttransplant,” Blood, 130:2257-64, 2017.
- C. Sipe et al., “Correction of Fanconi anemia mutations using digital genome engineering,” Int J Mol Sci, 23:1-20, 2022.
- S.M. Siegner et al., “Adenine base editing is an efficient approach to restore function in FA patient cells without double-stranded DNA breaks,” 489197, preprint on bioRxiv, 2022.