Researchers compare gene therapies for sickle cell disease in mice
Some approaches showed stronger stem cell persistence than CRISPR
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In a mouse study, blood stem cells from sickle cell disease (SCD) patients modified with CRISPR/Cas9 gene editing showed reduced long-term contribution compared with cells treated using other gene-based approaches.
“While all methods showed therapeutic potential, base editing and [gene therapy] provided superior outcomes over CRISPR-Cas9-mediated editing in a competitive [mice] transplantation model,” the researchers wrote.
The study, “Comparative Analysis of CRISPR-Cas9, lentiviral transduction, and base editing for sickle cell disease in a murine model,” was published in Blood Advances.
How sickle cell disease affects red blood cells
In SCD, a single alteration in the HBB gene leads to the production of hemoglobin S, or HbS — a faulty version of adult hemoglobin, the protein in red blood cells that carries oxygen throughout the body.
HbS tends to clump together, causing red blood cells to take on a sickle-like shape. These misshapen cells can break apart more easily or become stuck in small blood vessels, triggering painful episodes known as vaso-occlusive crises, along with other SCD symptoms.
Gene and gene-editing therapies have emerged as treatment options for certain genetic diseases, including SCD. Two such therapies are currently approved for SCD: Lyfgenia (lovotibeglogene autotemcel), a gene therapy, and Casgevy (exagamglogene autotemcel), a gene-editing therapy.
Lyfgenia uses a lentivirus carrier to deliver a modified HBB gene that produces HbAT87Q, a form of hemoglobin designed to reduce sickling. Casgevy uses CRISPR/Cas9 gene editing to target the BCL11A enhancer and reactivate production of fetal hemoglobin, a type of hemoglobin normally made before birth that is less prone to sickling.
Another approach under investigation is adenine base editing, which can alter a single DNA building block to convert HbS to HbG. Also known as Makassar hemoglobin, HbG is a naturally occurring variant believed to have anti-sickling properties.
Testing gene therapy approaches side by side
In this study, U.S.-based researchers used mice to directly compare three gene-based approaches — gene therapy, gene editing, and base editing — for SCD.
The team collected hematopoietic stem cells (HSCs), or blood cell precursor cells, from people with SCD and exposed them to each treatment. The treated cells were then transplanted into immunocompromised mice, whose weakened immune systems prevent rejection of the human cells.
Non-edited cells served as a control group. In some experiments, mice received an equal mixture of treated cells, allowing the different approaches to compete side by side in the same animal.
Initial experiments showed similar levels of engraftment — meaning the transplanted HSCs successfully took hold in the mice — across the three treatment groups, ranging from 35% to 39%.
About four months after transplantation, the intended genetic changes were detected in 95.8% of blood cells in the CRISPR/Cas9 group and 62.8% in the base-editing group. In the competitive setting, these levels dropped to 26.2% for base editing and 7.8% for CRISPR/Cas9 editing.
Our findings highlight the therapeutic potential of multiple SCD gene therapies while revealing differences in stem cell persistence and lineage dynamics that should guide protocol refinement.
Using the lentivirus gene therapy, fewer modified gene copies were detected in HSCs in the competitive setting.
When the researchers examined the animals’ bone marrow, high levels of HSC engraftment, above 80%, were seen across all three groups. Even in the competitive setting, engraftment reached about 75%.
The researchers also observed a higher proportion of CD33-positive myeloid cells in mice receiving base editing or gene therapy (about 25.8%-29.8%), compared with controls and CRISPR/Cas9 gene editing (about 5.8%-12.3%).
When looking at hemoglobin production, CRISPR/Cas9 editing alone led to a marked increase in red blood cells producing fetal hemoglobin (93.5%). However, this proportion was lower in the competitive setting (34.9%).
Levels of HbAT87Q reached 39% with gene therapy alone and 19.3% in the mixed group. HbG levels reached 73.3% with base editing alone and 39.3% in the mixed group.
How the treatments affected red blood cell sickling
In anti-sickling tests, red blood cell sickling was reduced by about 50% to 64.7% in the gene therapy, base-editing, and mixed treatment groups. CRISPR/Cas9 editing was significantly less effective, resulting in a 29.7% reduction in sickling.
To assess the long-term durability and repopulating capacity of gene-modified stem cells, the team performed a secondary transplant into new mice. Although overall engraftment levels were lower, the results mirrored those seen in the primary transplant, with base editing and gene therapy showing stronger persistence than CRISPR/Cas9 editing.
The researchers suggested that the reduced contribution of CRISPR/Cas9-edited cells may stem from the double-strand DNA breaks (DSBs) created by the technique. These breaks can trigger a DNA damage response, which may lead to cell death. Base editing avoids DSBs, helping preserve stem cell fitness.
“Our findings highlight the therapeutic potential of multiple SCD gene therapies while revealing differences in stem cell persistence and lineage dynamics that should guide protocol refinement,” the researchers wrote. “While preclinical mouse models offer mechanistic insights, only clinically validated approaches … directly inform patient care.”
