Prime editing corrected a disease-causing GATA2 mutation in patient-derived blood stem cells, increasing the proportion of functional gene copies from 50 to 77 percent in a preclinical study.
The approach achieved editing efficiencies of up to 70 percent without introducing the DNA double-strand breaks used in conventional CRISPR editing. The edited cells also showed improved engraftment after transplantation into immunodeficient mice, according to the study in Science Translational Medicine.
GATA2 deficiency is a rare inherited disorder caused by mutations affecting one copy of the GATA2 gene, which regulates blood stem cell maintenance and immune cell production. Patients may experience severe infections, bone marrow failure, and an increased risk of myeloid cancers. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment, but it carries risks including graft-versus-host disease and graft failure.
The Aarhus University team investigated whether prime editing could repair a seven-base-pair deletion in GATA2 directly in a patient’s CD34-positive hematopoietic stem and progenitor cells. Unlike conventional CRISPR-Cas9 editing, prime editing uses a modified Cas9 protein and a reverse transcriptase to rewrite a targeted DNA sequence without cutting both strands of the DNA molecule.
“We have shown that it’s possible to repair the genetic defect directly in a patient’s own stem cells,” said co-author Jonas Holst Wolff in a press release. “This brings us one step closer to a treatment that addresses the underlying cause of the disease rather than simply treating its consequences.”
Following optimization, the researchers achieved more than 70 percent prime editing in CD34-positive cells while reporting limited effects on cell fitness. The proportion of functional GATA2 alleles rose from the expected baseline of 50 percent to as much as 77 percent, accompanied by increased GATA2 protein expression.
Prime-edited cells retained their capacity to form blood cell colonies and produced up to 2.73-fold greater engraftment than untreated cells when transplanted into immunodeficient mice. Editing was maintained in repopulating stem cells 16 weeks after transplantation.
The researchers detected limited unintended changes at the target site and no editing at the 20 highest-ranked predicted off-target sites. Shortening the period of stem cell stimulation before editing to less than 24 hours also reduced the DNA damage response and improved multilineage engraftment.
A complementary approach known as PASSIGE was used to insert a larger corrective GATA2 sequence that could potentially address more disease-causing variants. However, integration reached only 4.6 percent of alleles, indicating that further optimization will be required.
Newsletters
Receive the latest pharmaceutical news, personalities, education, and career development – weekly to your inbox.
